JP2019517797A - Semibiosynthetic production of fatty alcohols and fatty aldehydes - Google Patents

Abstract

The present invention relates to the semibiosynthetic production of fatty alcohols and fatty aldehydes. The present application relates to the acquisition or production of one or more unsaturated lipid moieties from a biological source, to one or more fatty alcohols and / or one or more fatty aldehydes. The present invention relates to a process for producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties by combining with the conversion by non-biological means of unsaturated lipid moieties of . The application also relates to a recombinant microorganism having a biosynthetic pathway for the production of one or more unsaturated lipid moieties. One or more fatty alcohols can be further chemically converted to one or more corresponding fatty acetic acids. One or more fatty alcohols, one or more fatty aldehydes and / or one or more fatty acetic acids produced by the methods described herein may be one or more insect pheromone, one or more There may be multiple flavors, one or more seasonings, or one or more polymer intermediates. [Selected figure] Figure 1

Description

This application claims the benefit of priority of US Provisional Patent Application No. 62 / 346,335, filed Jun. 6, 2016, which is incorporated herein by reference in its entirety Be

[0002] A sequence listing related to the present application is provided in text form instead of a paper copy, and is incorporated herein by reference. The name of the text file containing the sequence listing is PRVI-017_01 WO_SeqList_ST25.txt. The text file is about 103 KB, is created on June 5, 2017, and is submitted electronically via EFS-Web.

[0003] The application includes biosynthetic pathways for obtaining one or more unsaturated lipid moieties from one or more naturally occurring organisms or for producing one or more unsaturated lipid moieties. Produce one or more unsaturated lipid moieties in one or more recombinant microorganisms engineered as such, and one or more unsaturated lipid moieties with one or more free fatty acids (FFAs) and / or Chemically convert to one or more fatty acid alkyl ester (FAAE), reduce one or more FFAs and / or one or more FAAEs, reduce by reduction one or more fatty alcohols and / or one By producing one or more fatty aldehydes, one or more fatty alcohols and / or one or more fatty alcohol moieties from one or more unsaturated lipid moieties. Other relates to a process for producing one or more fatty aldehydes. One or more fatty alcohols can be further chemically converted to one or more corresponding fatty acetates. One or more fatty alcohols, one or more fatty aldehydes and / or one or more fatty acetic acids may be useful as insect pheromone, perfumes, flavors and polymer intermediates. The application further relates to a composition comprising a recombinant microorganism useful in the biosynthesis of unsaturated lipid moieties and one or more fatty alcohols and / or one or more fatty aldehydes.

[0004] As global food demand increases, the demand for effective pest control is increasing. Conventional insecticides are one of the most widely used chemical repellents because they are readily available, fast-acting and reliable. However, due to the excessive use, misuse and abuse of these chemicals, resistant pests have emerged, the natural ecological environment has changed, and in some cases environmental destruction has resulted.

[0005] The use of insect pheromone to control pest populations has become increasingly popular as a viable, safe and environmentally friendly alternative to traditional insecticides. Since their discovery in the late 1950's, these molecules have been shown to be effective in reducing insect populations through a variety of methods, including mass capture, induced killing, and mating disruption. The last method is in particular a non-toxic means of pest control and exploits the ability of synthetic pheromone to shield the naturally occurring pheromone thereby causing confusion and distraction.

[0006] Although pheromone has significant potential in agricultural insect control, the cost of synthesizing pheromone using currently available techniques is very high, so extensive use of this sustainable technology except for high value crops Is hampered. Thus, there is an existing need to develop new technologies for cost-effective production of insect pheromone and related flavors, flavors and polymeric intermediates. We address this need by developing a method for the semi-biosynthetic production of fatty alcohols and / or fatty aldehydes comprising synthetic insect pheromone from low cost feedstocks.

[0007] The present application relates to the acquisition or production of one or more unsaturated lipid moieties from a biological source into one or more fatty alcohols and / or one or more fatty aldehydes. The invention relates to a method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties by combining with the conversion of unsaturated lipid moieties by non-biological means. The application also relates to a recombinant microorganism having a biosynthetic pathway for the production of one or more unsaturated lipid moieties. The one or more fatty alcohols and / or one or more fatty aldehydes produced by the methods described herein can be one or more insect pheromone, one or more fragrances, one or more Or one or more polymer intermediates.

[0008] In one aspect, a method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is one or more naturally occurring organisms. Obtaining one or more unsaturated lipid moieties therefrom, as well as direct reduction of the one or more unsaturated lipid moieties and reduction to reduce one or more fatty alcohols and / or one or more fatty aldehydes Includes production steps.

[0009] In another aspect, the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is one or more naturally occurring Obtaining one or more unsaturated lipid moieties from the organism; chemically converting one or more unsaturated lipid moieties to one or more free fatty acids (FFAs); one or more FFAs Esterifying one or more fatty acid alkyl esters (FAAE); and reducing one or more FFAs and / or one or more FAAEs, wherein the reduction results in one or more fats Including the step of producing alcohol and / or one or more fatty aldehydes. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[0010] In another aspect, the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is one or more naturally occurring Obtaining one or more unsaturated lipid moieties from the organism; chemically converting one or more unsaturated lipid moieties to one or more FAAEs; and reducing one or more FAAEs The process comprises the process of producing one or more fatty alcohols and / or one or more fatty aldehydes by reduction. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[0011] In one embodiment, the one or more naturally occurring organisms are insects. In another embodiment, the one or more naturally occurring organisms are not insects. In some embodiments, one or more naturally occurring organisms are selected from the group consisting of plants, algae and bacteria. In some embodiments, the plant is from a family selected from the group consisting of pokeweed family and buckthorn family. In a particular embodiment, the plant is Gevuina, Calendula, Limnanthes, Lunaria, Carum, Daucus, Coriandrum, Macadamia, Myristica, Licania, Aleurites, Ricinus, Corylus, Kermadecia. And species from the genera Asclepias, Cardwellia, Grevillea, Orites, Ziziphus, Hicksbeachia, Hippophae, Epopdra, Plasospermum, Xylomelum and / or Simmondsia. In some embodiments, the plants are Gevuina avellana, Corylus avellana, Kermadecia sinuata, Asclepias syriaca, Cardwellia sublimis, Grevillea exul var. and species from Rubiginosa, Orites diversifolius, Orites revoluta, Ziziphus jujube, Hicksbeachia pinnatifolia, and Grevillea decora. In some embodiments, the algae comprises green algae. In certain embodiments, the green algae comprises a species from the genus Pediastrum. In some embodiments, the green algae comprises Pediastrum simplex. In some embodiments, the bacteria comprises gram negative bacteria. In some embodiments, the gram negative bacteria comprises a species from the genus Myxococcus. In certain embodiments, the gram negative bacteria comprises Myxococcus xanthus. In some embodiments, the bacteria comprises cyanobacteria. In some embodiments, the cyanobacteria comprises a species from the genus Synechococcus. In certain embodiments, the cyanobacteria comprises Synechococcus elongatus.

[0012] In another aspect, a method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties comprises one or more unsaturated lipid moieties Producing one or more unsaturated lipid moieties in one or more recombinant microorganisms engineered to include a biosynthetic pathway for the production of a; one or more unsaturated lipid moieties; Chemically converting to or more free fatty acids (FFA) and / or one or more fatty acid methyl esters (FAAE); and reducing one or more FFAs and / or one or more FAAE Including the step of reducing to produce one or more fatty alcohols and / or one or more fatty aldehydes. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[0013] In one embodiment, one or more recombinant microorganisms use one or more carbon sources for the production of one or more unsaturated lipid moieties. In another embodiment, the one or more carbon sources include sugars, glycerol, ethanol, organic acids, alkanes, and / or fatty acids. In certain embodiments, one or more carbon sources are selected from the group consisting of alkanes and fatty acids. In some embodiments, one or more recombinant microorganisms are highly resistant to alkanes and / or fatty acids. In some embodiments, one or more recombinant microorganisms accumulate lipids. In other embodiments, one or more recombinant microorganisms comprise one or more bacteria and / or fungi. In certain embodiments, the one or more fungi are oleaginous yeast. In one embodiment, the oleaginous yeast comprises species from the genera Yarrowia and / or Candida. In another embodiment, the oleaginous yeast comprises Yarrowia lipolytica and / or Candida viswanathii / tropicalis.

[0014] In one embodiment, one or more recombinant microorganisms engineered to include a biosynthetic pathway for the production of one or more unsaturated lipid moieties comprises one or more desaturases, 1 One or more acyl CoA oxidases, one or more acyl glycerol lipases, one or more flavoprotein pyridine nucleotide cytochrome reductase, one or more elongases, one or more thioesterases, one or more One or more selected from glycerol-3-phosphate acyltransferase, one or more lysophosphatidic acid acyltransferases, one or more diacylglycerol acyltransferases, and / or one or more lipid binding proteins It includes a plurality of exogenous biosynthetic enzyme. In another embodiment, the one or more desaturases are selected from the group consisting of fatty acyl CoA desaturases and fatty acyl ACP desaturases. In some embodiments, one or more flavoprotein pyridine nucleotide cytochrome reductase is selected from the group consisting of cytochrome b5 reductase and NADPH-dependent cytochrome P450 reductase. In some embodiments, the one or more elongases are selected from the group consisting of ELO1, ELO2 and ELO3, or any combination thereof. In some embodiments, one or more thioesterases are selected from the group consisting of acyl ACP thioesterases and acyl CoA thioesterases. In some embodiments, the one or more glycerol-3-phosphate acyltransferase is dual glycerol-3-phosphate O-acyltransferase / dihydroxyacetone phosphate acyltransferase. In some embodiments, one or more lysophosphatidic acid acyltransferases are selected from the group consisting of SLC1, ALE1 and LOA1, or any combination thereof. In some embodiments, one or more diacylglycerol acyltransferases are selected from the group consisting of acyl CoA-dependent acyltransferases and phospholipids: diacylglycerol acyltransferases. In some embodiments, one or more lipid binding proteins are sterol carrier protein 2 (SCP2). In some embodiments, one or more of the exogenous biosynthetic enzymes are Saccharomyces cerevisiae, Yarrowia, Candida, Helicoverpa, Agrotis, Trichoplusia, Spodoptera, Ostrinia, Amyelois, Lobesia, Cydia, Grapholita, Lampronia, Sesamia, Plodia, Bombyx , Phoenix, Rattus, Arabidopsis, Plutella and Bicyclus are from a genus selected from the group consisting of In some embodiments, the one or more exogenous biosynthetic enzymes are Saccharomyces cerevisiae, Yarrowia lipolytica, Candida albicans, Candida tropicalis, Candida viswanathii, Helicoverpa armigera, Helicoverpa zea, Agrotis segetum, S. exigua, Amyelois transitella, Lobesia botrana, Cydia pomonella, Lampronia capitella, Grapholita molesta, Ostrin a scapulalis, Ostrinia furnacalis, Ostrinia nubilalis, Ostrinia latipennis, Ostrinia ovalipennis, Plodia interpunctella, Sesamia inferens, Bombyx mori, Phoenix dactylifera, Rattus norvegicus, Arabidopsis thaliana, is derived from a genus selected from the group consisting of Plutella xylostella and Bicyclus anynana.

[0015] In some embodiments, the fatty acyl desaturases are: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. Or a desaturase that can utilize fatty acyl CoA as a substrate with a chain length of 24 carbon atoms.

[0016] In some embodiments, the fatty acyl desaturase is a fatty acid or a derivative thereof such as, for example, fatty acid CoA ester at a position of C5, C6, C7, C8, C9, C9, C10, C11, C12, or C13. Double bonds can be made.

[0017] In one exemplary embodiment, the fatty acyl desaturase is a Z11 desaturase. In various embodiments described herein, the Z11 desaturase, or the nucleic acid sequence encoding it, is of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or It is possible to isolate from organisms of An additional Z11 desaturase, or a nucleic acid sequence encoding it, can be isolated from Bombyx mori, Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphania nitidalis. In an exemplary embodiment, the Z11 desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787, and AY493438. In some embodiments, the nucleic acid sequence encoding the Z11 desaturase from the organism of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized . In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NO: 3, 11, 17 and 19 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 25 from Trichoplusia ni. In another embodiment, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOS: 4 and 9 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 29 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 5 and 16 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 26 and 27 from Thalassiosira pseudonana. In a particular embodiment, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NO: 6, 10 and 23 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 28 from Amyelois transitella. In a further embodiment, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 7, 12, 18, 20 and 24 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 1 from Helicoverpa zea. In some embodiments, the Z11 desaturase is S. coli. It comprises the amino acid sequence shown in SEQ ID NO: 2 from inferens. In some embodiments, the Z11 desaturases are GenBank Accession Nos. AF416738, AGH12217.1, AII 21943.1, CAJ43430.2, AF441221, AAF 81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, It includes the amino acid sequences set forth in ABX71630.1 and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, the complete or partial Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of the Z11 desaturase has been replaced with an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 8, 21 and 22. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 36, 37 and 38.

[0018] In a specific embodiment, the Z11 desaturase is Z11-13: acyl CoA, E11-13: acyl CoA, (Z, Z) -7, 11-13: acyl CoA, Z11-14: acyl CoA, E11-14: Acyl-CoA, (E, E) -9, 11-14: Acyl-CoA, (E, Z) -9, 11-14: Acyl-CoA, (Z, E) -9, 11-14: Acyl CoA, (Z, Z) -9, 11-14: Acyl CoA, (E, Z) -9, 11-15: Acyl CoA, (Z, Z) -9, 11-15: Acyl CoA, Z11-16 : Acyl CoA, E11-16: Acyl CoA, (E, Z) -6, 11-16: Acyl CoA, (E, Z) -7, 11-16: Acyl CoA, (E, Z) -8, 11 -16: acyl CoA, (E, E) -9, 11-1 : Acyl CoA, (E, Z) -9, 11-16: Acyl CoA, (Z, E) -9, 11-16: Acyl CoA, (Z, Z) -9, 11-16: Acyl CoA, ((E) E, E) -11, 13-16: acyl CoA, (E, Z) -11, 13-16: acyl CoA, (Z, E) -11, 13-16: acyl CoA, (Z, Z)- 11, 13-16: Acyl-CoA, (Z, E) -11, 14-16: Acyl-CoA, (E, E, Z) -4, 6, 11-16: Acyl-CoA, (Z, Z, E) -7, 11, 13-16: Acyl CoA, (E, E, Z, Z) -4, 6, 11, 13-16: Acyl CoA, Z11-17: Acyl CoA, (Z, Z) -8, 11-17: Acyl CoA, Z11-18: Acyl CoA, E11-18: Acyl CoA, (Z, Z) -11 , 13-18: catalyzes the conversion of fatty acyl-CoA to mono- or polyunsaturated products selected from acyl-CoA, (E, E) -11, 14-18: acyl-CoA or combinations thereof.

[0019] In another exemplary embodiment, the fatty acyl desaturase is a Z9 desaturase. In various embodiments described herein, the Z9 desaturase, or a nucleic acid sequence encoding it, is isolated from an organism of the species Ostrinia furnacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, or Helicoverpa zea It is possible. In an exemplary embodiment, the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332, AF482906, and AAF81788. In some embodiments, the nucleic acid sequence encoding the Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 13 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 30 from Ostrinia furnacalis. In another embodiment, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 14 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 31 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 15 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 32 from Helicoverpa zea.

[0020] In a specific embodiment, the Z9 desaturase is Z9-11: acyl CoA, Z9-12: acyl CoA, E9-12: acyl CoA, (E, E) -7, 9-12: acyl CoA, (E, Z) -7, 9-12: Acyl CoA, (Z, E) -7, 9-12: Acyl CoA, (Z, Z) -7, 9-12: Acyl CoA, Z9-13: Acyl CoA, E9-13: Acyl CoA, (E, Z) -5, 9-13: Acyl CoA, (Z, E) -5, 9-13: Acyl CoA, (Z, Z) -5, 9-13 : Acyl-CoA, Z9-14: Acyl-CoA, E9-14: Acyl-CoA, (E, Z) -4, 9-14: Acyl-CoA, (E, E) -9, 11-14: Acyl-CoA, (E , Z) -9, 11-14: acyl CoA, (Z, E) -9, 11-14: Syl-CoA, (Z, Z) -9, 11-14: Acyl-CoA, (E, E) -9, 12-14: Acyl-CoA, (Z, E) -9, 12-14: Acyl-CoA, (Z , Z) -9, 12-14: Acyl-CoA, Z9-15: Acyl-CoA, E9-15: Acyl-CoA, (Z, Z) -6, 9-15: Acyl-CoA, Z9-16: Acyl-CoA, E9 -16: Acyl-CoA, (E, E) -9, 11-16: Acyl-CoA, (E, Z) -9, 11-16: Acyl-CoA, (Z, E) -9, 11-16: Acyl-CoA , (Z, Z) -9, 11-16: Acyl-CoA, Z9-17: Acyl-CoA, E9-18: Acyl-CoA, Z9-18: Acyl-CoA, (E, E) -5, 9-18: Acyl CoA, (E, E) -9, 12-18: Acyl CoA, (Z, Z) -9 , 12-18: Acyl-CoA, (Z, Z, Z) -3, 6, 9-18: Acyl-CoA, (E, E, E) -9, 12, 15-18: Acyl-CoA, (Z, Z) , Z) -9, 12, 15-18: Catalyzing the conversion of fatty acyl-CoA into mono- or polyunsaturated products selected from acyl-CoA or combinations thereof.

[0021] In some embodiments, the fatty acyl desaturase is E5-10: acyl CoA, E7-12: acyl CoA, E9-14: acyl CoA, E11-16: acyl CoA, E13-18: Acyl-CoA , Z7-12: Acyl CoA, Z9-14: Acyl CoA, Z11-16: Acyl CoA, Z13-18: Acyl CoA, Z8-12: Acyl CoA, Z10-14: Acyl CoA, Z12-16: Acyl CoA, Z14-18: Acyl CoA, Z7-10: Acyl CoA, Z9-12: Acyl CoA, Z11-14: Acyl CoA, Z13-16: Acyl CoA, Z15-18: Acyl CoA, E7-10: Acyl CoA, E9 -12: Acyl CoA, E11-14: Acyl CoA, E13-16: Acyl CoA, E15-18 Acyl-CoA, E5Z7-12: Acyl-CoA, E7Z9-12: Acyl-CoA, E9Z11-14: Acyl-CoA, E11Z13-16: Acyl-CoA, E13Z15-18: Acyl-CoA, E6E8-10: Acyl-CoA, E8E10-12: Acyl CoA, E10 E 12-14: Acyl CoA, E 12 E 14-16: Acyl-CoA, Z 5 E 8-10: Acyl CoA, Z 7 E 10-12: Acyl CoA, Z 9 E 12-14: Acyl CoA, Z 11 E 14-16: Acyl CoA, Z 13 E 16-18: Acyl CoA, Z3-10: Acyl CoA, Z5-12: Acyl CoA, Z7-14: Acyl CoA, Z9-16: Acyl CoA, Z11-18: Acyl-CoA, Z3 Z5-10: Acyl CoA, Z5 Z7-12: Acyl CoA, Z7Z9-14: catalyzes the conversion of fatty acyl-CoA to a mono- or polyunsaturated intermediate selected from acyl-CoA, Z9Z11-16: acyl-CoA, Z11Z13-16: acyl-CoA and Z13Z15-18: acyl-CoA.

[0022] In some embodiments, the recombinant microorganism can express a bifunctional desaturase capable of catalyzing the subsequent desaturation of the two double bonds.

[0023] In some embodiments, the recombinant microorganism is a saturated _C 6 _{-C 24} corresponding mono- or polyunsaturated _C 6 _{-C 24} fatty acyl desaturase that catalyzes the conversion of fatty acyl CoA fatty acyl CoA And more than one exogenous nucleic acid molecule encoding. For example, the recombinant microorganism can express an exogenous nucleic acid molecule encoding a Z11 desaturase and another exogenous nucleic acid molecule encoding a Z9 desaturase.

[0024] In some embodiments, one or more recombinant microorganisms engineered to include a biosynthetic pathway for the production of one or more unsaturated lipid moieties have a given cycle producing a starting one or polyunsaturated _C 6 _{-C 24} primary or having two carbon cut as compared to fatty acyl-CoA substrate polyunsaturated _C 4 _{-C 22} fatty acyl-CoA intermediates in the cycle, acyl CoA Acyl-CoA oxidase which catalyzes the conversion of mono- or polyunsaturated C ₆ -C ₂₄ fatty acyl-CoA into a truncated mono- or polyunsaturated fatty acyl CoA after one or more successive cycles of oxidase activity And at least one exogenous nucleic acid molecule that encodes. In some embodiments, the acyl CoA oxidase is selected from Table 3a.

[0025] In some embodiments, the recombinant microorganism is preferably an acylglycerol lipase and / or sterol ester ester to hydrolyze ester bonds of> C16,> C14,> C12 or> C10 acylglycerol substrates. It further comprises at least one endogenous or exogenous nucleic acid molecule encoding an esterase enzyme. In some embodiments, the expressed acylglycerol lipase and / or sterol ester esterase enzyme is selected from Table 3b.

[0026] In some embodiments, one or more recombinant microorganisms engineered to include a biosynthetic pathway for the production of one or more unsaturated lipid moieties comprises one or more Further engineered to lack, destroy, mutate, and / or reduce the activity of one or more endogenous proteins in a pathway that competes with the biosynthetic pathway for the production of saturated lipid moieties There is. In some embodiments, the one or more endogenous proteins are from a genus selected from the group consisting of Saccharomyces, Yarrowia and Candida. In another embodiment, the one or more endogenous proteins are from a species selected from the group consisting of Saccharomyces cerevisiae, Yarrowia lipolytica, Candida albicans and Candida tropicalis / Candida viswanathii.

[0027] In some embodiments, the recombinant microorganism lacks, destroys, mutates, and / or one or more endogenous proteins involved in one or more undesired fatty acid elongation pathways. And / or is manipulated to reduce its activity. In certain embodiments, one or more endogenous proteins involved in one or more undesired fatty acid elongation pathways are β-ketoacyl CoA reductase, β-hydroxyacyl CoA dehydratase, enoyl CoA reductase Or any combination of these.

[0028] In another embodiment, the recombinant microorganism is a mutation that lacks, destroys, one or more endogenous proteins involved in one or more fat body storage and / or recycling pathways. And / or are engineered to reduce its activity. In certain embodiments, one or more endogenous proteins involved in one or more fat body storage and / or recycling pathways are triacylglycerol lipase, lysophosphatidic acid acyltransferase, steryl ester hydrolase , Acyl CoA synthetase, or any combination thereof. In some embodiments, the triacylglycerol lipase that is missing, destroyed, mutated and / or whose activity is reduced is YALI0D 17534 g (TGL3). In some embodiments, the fatty acyl CoA synthetase (fatty acid transporter) that is missing, destroyed, mutated and / or whose activity is reduced is YALI0E16016 g (FAT1).

[0029] In some embodiments, the recombinant microorganism lacks, destroys, mutates and / or / or one or more endogenous proteins involved in one or more fatty acid degradation pathways. It is manipulated to reduce its activity. In certain embodiments, the fatty acid degradation pathway is a beta oxidation pathway. In certain embodiments, the one or more endogenous proteins are selected from the group consisting of acyl CoA oxidase, multifunctional enoyl CoA hydratase-β-hydroxyacyl CoA dehydrogenase, β-ketoacyl CoA thiolase, or any combination thereof It is selected. In some preferred embodiments, one or more genes of the microbial host encoding the acyl CoA oxidase are absent to eliminate or reduce cleavage of the desired fatty acyl CoA beyond the desired chain length. Or down-regulated. In some embodiments, the recombinant microorganism is Y.1. lipolytica POX1 (YALI0E32835 g), Y.1. lipolytica POX2 (YALI0F10857g), Y.1. lipolytica POX3 (YALI0D24750 g), Y.1. lipolytica POX 4 (YALI0E27654 g), Y.1. lipolytica POX5 (YALI0 C23859 g), Y.1. lipolytica POX 6 (YALI0E06567 g); S. cerevisiae POX1 (YGL205W); Candida POX2 (CaO 19.1655, CaO 19.9224, CTRG_02374, M18259), Candida POX4 (CaO 19.1652, CaO 19.9221, CTRG_02377, M12160), and Candida POX5 (CaO 19.5723, CaO19.13, 146) CTRG — 02721, including deletion, disruption, mutation, and / or reduction of its activity of one or more endogenous acyl-CoA oxidase enzymes selected from the group consisting of

[0030] In some embodiments, glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerol phospholipid acyltransferase (GPLAT) and / or diacylglycerol acyltransferase (DGAT) One or more genes of the encoding recombinant microorganism are missing or down-regulated and replaced by one or more heterologous GPAT, LPAAT, GPLAT, or DGAT variants. In some embodiments, the one or more acyltransferase variants are derived from one or more heterologous acyltransferases selected from Table 3c. In some embodiments, the recombinant microorganism is Y.1. lipolytica YALI0 C00209 g, Y. lipolytica YALI 0 E 18964 g, Y. lipolytica YALI 0 F 19514 g, Y. lipolytica YALI 0 C 14014 g, Y. lipolytica YALI 0 E 16 797 g, Y. lipolytica YALI 0 E 327 69 g and Y. lipolytica YALI 0 D 0 79 86 g, S. S. cerevisiae YBL011 w, S.I. S. cerevisiae YDL052 c, S.I. S. cerevisiae YOR 175 C, S.I. S. cerevisiae YPR139C, S.I. S. cerevisiae YNR 008 w and S. S. cerevisiae YOR245c, as well as Candida I 503_02577, Candida CTRG_02630, Candida CaO 19. 250, Candida CaO 19. 7881, Candida CTRG _ 02437, Candida CaO 19. 1881, Candida Ca O 19 9.9 43, Candida CT R O 018 47, Candida Ca O 19 ci. Selected from the group consisting of CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203 and Candida CTRG_06209 Missing one or more endogenous acyltransferase enzyme comprises disruption, mutation, and / or a reduction in its activity.

[0031] In certain embodiments, the fatty acid degradation pathway is an omega oxidation pathway. In certain embodiments, one or more endogenous proteins are selected from the group consisting of monooxygenase (CYP 52), fatty alcohol oxidase, fatty alcohol dehydrogenase, alcohol dehydrogenase, fatty aldehyde dehydrogenase, or any combination thereof . In some preferred embodiments, the recombinant microorganism is Y.1. lipolytica YALI0 E25982 g (ALK1), Y.1. lipolytica YALI0 F01320 g (ALK2), Y.1. lipolytica YALI0 E23474 g (ALK3), Y.1. lipolytica YALI0 B13816 g (ALK4), Y.1. lipolytica YALI0B 13838 g (ALK5), Y.1. lipolytica YALI 0 B 01 848 g (ALK 6), Y. lipolytica YALI0A 15488 g (ALK7), Y.1. lipolytica a YALI 0 C 12 122 g (ALK 8), Y. lipolytica YALI0B06248 g (ALK9), Y.1. lipolytica YALI 0 B 20 702 g (ALK 10), Y. lipolytica YALI 0 C 10054 g (ALK 11) and Y. and L. loss, destruction, mutation, and / or reduction of the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI 0 A 20130 g (ALK 12).

[0032] In another embodiment, the recombinant microorganism is designed to deplete, destroy, mutate, and / or reduce the activity of one or more endogenous proteins involved in peroxisome biogenesis It is operated by. In certain embodiments, one or more endogenous proteins involved in peroxisomal biogenesis are PEX1, PEX2, PEX3, PEX4, PEX5, PEX7, PEX7, PEX8, PEX10, PEX11, PEX12, PEX13, PEX14. , PEX 15, PEX 17, PEX 18, PEX 21, PEX 22, PEX 25, PEX 27, PEX 28, PEX 29, PEX 30, PEX 31, PEX 32, PEX 32, or any combination thereof.

[0033] In another embodiment, the one or more recombinant microorganisms are further engineered to increase intracellular levels of coenzyme. In certain embodiments, the coenzyme is NADH and / or NADPH. In some embodiments, one or more recombinant microorganisms are glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase, transketolase, ribose-5 -Consists of ketol isomerase, D-ribulose-5-phosphate 3-epimerase, NADP + dependent isocitrate dehydrogenase, NAD + or NADP + dependent malate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, transhydrogenase or any combination thereof It is further engineered to express one or more endogenous or exogenous proteins selected from the group. In another embodiment, expression of one or more endogenous or exogenous proteins is linked to complementation of a co-substrate. In some embodiments, one or more recombinant microorganisms are glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase, transketolase, ribose-5 -Lacking, disrupting, mutating, one or more endogenous proteins selected from the group consisting of ketol isomerase, phosphate, D-ribulose-5-phosphate 3-epimerase, or any combination thereof And / or is further manipulated to reduce its activity.

[0034] In some embodiments, the recombinant microorganism expresses one or more acyl-CoA oxidase enzymes and lacks, destroys, mutates, and lacks one or more endogenous acyl-CoA oxidase enzymes And / or is manipulated to reduce its activity. In some embodiments, the one or more acyl CoA oxidase enzymes being expressed are different from the one or more endogenous acyl CoA oxidase enzymes that are missing or downregulated. In other embodiments, the one or more acyl CoA oxidase enzymes being expressed modulate the chain length of one or more unsaturated lipid moieties. In another embodiment, the one or more acyl CoA oxidase enzymes being expressed are selected from Table 3a.

[0035] In another aspect, the recombinant microorganism used in any of the methods for producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is It is disclosed herein.

[0036] In another aspect, the recombinant microorganism used in any of the methods for producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties Methods of making are disclosed herein.

[0037] In one embodiment, the one or more unsaturated lipid moieties are (Z) -3-hexenoic acid, (Z) -3-nonenoic acid, (Z) -5-decenoic acid, (E)- 5-decenoic acid, (Z) -7-dodecenoic acid, (E) -8-dodecenoic acid, (Z) -8-dodecenoic acid, (Z) -9-dodecenoic acid, (E, E) -8, 10 -Dodecadienoic acid (dodecadienoic acid), (7E, 9Z)-dodecadienoic acid, (Z) -9-tetradecenoic acid (tetradecenoic acid), (Z) -11-tetradecenoic acid, (E) -11-tetradecenoic acid, (Z ) -7-Hexadecenoic acid, (Z) -9-hexadecenoic acid, (Z) -11-hexadecenoic acid, (Z, Z) -11, 13-hexadecadienoic acid, (11 Z, 13 E)- Hexadecadienoic acid, (9Z, 11E) -hexadecadienoic acid, (Z) -1 It is selected from 3-octadecenoic acid, (Z, Z, Z, Z, Z) -3,6,9,12,15-tricosapentaenoic acid, or any combination thereof. In another embodiment, the one or more unsaturated lipid moieties are (Z) -9-tetradecenoic acid (Z9-14: COOH), (Z) -11-hexadecenoic acid (Z11-16: COOH) and Z) selected from the group consisting of 11-octadecenoic acid (Z11-18: COOH). In another embodiment, the one or more fatty alcohols and / or one or more fatty aldehydes comprise one or more insect pheromone, a fragrance, a flavor and / or a polymeric intermediate. In certain embodiments, one or more fatty alcohols and / or one or more fatty aldehydes comprise one or more insect pheromone.

[0038] An exemplary insect pheromone in the form of a fatty alcohol, fatty aldehyde, or fatty acetic acid that can be made using the recombinant microorganisms and methods described herein is (Z) -11- Hexadecenal, (Z) -11 hexadecenyl acetate, (Z) -9-tetradecenyl acetate, (Z, Z) -11, 13-hexadecadienal, (9 Z, 11 E) -hexadecadienal , (E, E) -8,10-dodecadien-1-ol, (7E, 9Z) -dodecadienyl acetate, (Z) -3-nonen-1-ol, (Z) -5-decene-1-l. Or (Z) -5-decenyl acetate, (E) -5-decene-1-ol, (E) -5-decenyl acetate, (Z) -7-dodecene-1-ol, (Z )-7-dodecenyl acetate, (E -8- dodecen-1-ol, (E) -8-dodecenyl acetate, (Z) -8-dodecen-1-ol, (Z) -8-dodecenyl acetate, (Z) -9 -Dodecen-1-ol, (Z) -9-dodecenyl acetate, (Z) -9-tetradecen-1-ol, (Z) -11-tetradecen-1-ol, (Z) -11-tetra Decenyl acetate, (E) -11-tetradecen-1-ol, (E) -11-tetradecenyl acetate, (Z) -7-hexadecen-1-ol, (Z) -7-hexadecenal, Z) -9-hexadecen-1-ol, (Z) -9-hexadecenal, (Z) -9-hexadecenyl acetate, (Z) -11-hexadecen-1-ol, (Z) -13-octadecenene -1-ol and (Z) -13-octadecenal Including, but not limited to.

[0039] Any of the methods for producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein comprising a chemical conversion step In one embodiment, chemically converting the one or more unsaturated lipid moieties to one or more FFAs and / or one or more FAAEs comprises one or more unsaturated lipid moieties. Isolating, optionally enriching one or more unsaturated lipid moieties having a specific chain length, and treating one or more unsaturated lipid moieties, comprising fatty acids The method further comprises the step of producing a derivative. In some embodiments, the step of treating one or more unsaturated lipid moieties comprises saponification of one or more unsaturated lipid moieties to one or more FFAs. In some embodiments, treating the one or more unsaturated lipid moieties comprises esterification of the one or more unsaturated lipid moieties to one or more FAAEs. In one embodiment, the optional step of concentrating one or more unsaturated lipid moieties having a particular chain length comprises a separation method. In another embodiment, the separation method comprises distillation and / or chromatography. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[0040] In any one embodiment of the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein, The reduction of one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs to one or more fatty alcohols and / or one or more fatty aldehydes Contacting one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs with one or more stoichiometric reducing agents. In some embodiments, the one or more stoichiometric reducing agents are sodium bis (2-methoxyethoxy) aluminum hydride, Vitride (Red-Al, Vitride, SMEAH) and / or hydrogenated diisobutylaluminum (DIBAL) )including. In certain embodiments, the step of reducing one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs comprises bulky cyclic nitrogen Lewis to modify the reducing agent. Contains a base. In one embodiment, the bulky cyclic nitrogen Lewis base provides the selectivity of the reduction step for the production of one or more fatty aldehydes. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[0041] In any one embodiment of the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein, The step of reducing one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs to one or more fatty alcohols comprises one or more unsaturated lipid moieties, one Contacting one or more FFAs and / or one or more FAAEs with one or more transition metal catalysts. In certain embodiments, the one or more transition metal catalysts comprise one or more Group VIII catalysts. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[0042] In one embodiment of any of the methods of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein, The produced fatty alcohol is further oxidized to fatty aldehyde by the oxidation process. In some embodiments, the oxidation step comprises one or more partial oxidation methods. In certain embodiments, one or more partial oxidation methods include NaOCI / TEMPO. In another embodiment, the oxidation step comprises copper catalyzed aerobic oxidation.

[0043] In one embodiment of any one of the methods of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein, The produced one or more fatty alcohols are further chemically converted to one or more corresponding fatty acetate esters. In certain embodiments, chemically converting one or more fatty alcohols to one or more corresponding fatty acetate esters comprises contacting one or more fatty alcohols with acetic anhydride .

[0044] A method for producing one or more fatty alcohols, one or more fatty aldehydes and / or one or more fatty acetic acids from one or more unsaturated lipid moieties disclosed herein In any one embodiment, one or more fatty alcohols, one or more fatty aldehydes and / or one or more fatty acetic acids are further concentrated by a separation procedure.

[0045] In another aspect, from one or more unsaturated lipid moieties disclosed herein to one or more fatty alcohols, one or more fatty aldehydes and / or one or more fatty acetic acids One or more compositions produced by any of the methods of producing

[0046] Illustrative embodiments of the present disclosure are described in the following figures.

[0047] Figure 1 illustrates the concept of semi-biosynthetic production of fatty alcohols and fatty aldehydes. [0048] Figure 5 illustrates the conversion of saturated and unsaturated molecules to alcohols and aldehydes. [0049] Figure 5 illustrates the proposed biohydroxylation and fatty acyl CoA reduction route to (E) -5-decene. [0050] Figure 17 illustrates the production of 17 commodity oils from 1976 to 2016 forecast (Gunstone, FD. Vegetable Oils in Food Technology: Composition, Properties and Uses. Wiley-Blackwell, 2011). [0051] FIG. 1 shows an overlay of chromatograms of different vegetable oils. No. Only 5 (blue) chilli hazelnut oil peaks at a retention time of 6.2 minutes corresponding to methyl (Z) -hexadeca-11-enoate. [0052] Figure 5 shows a comparison of TMSH (top) transesterified chili hazelnut oil and biodiesel produced by alkaline methanolysis (bottom). The chromatograms appear to be similar, but with slight differences in apparent peak areas. [0053] FIG. 7 shows an overlay of GC-FID chromatograms of various fractions collected by single distillation. The first fraction (red) is collected at 55-82 ° C. and contains the largest fraction of C16 compound (about 6 minutes). The second fraction (green) was collected at 120-146 ° C. The majority of the material is the various C18 acids, but this fraction still shows significant amounts of C16. Fraction 3 (blue) contains seed residue. Although C16 is barely seen, most of the long chain homologs (about 9-11 minutes) are seen. [0054] Figure 5 shows the reduction of an ester to an aldehyde with a modified Red-Al and its preparation (Shin WK et al. (2014) Bull. Korean Chem. Soc. 35: 2169-2171). [0055] FIG. 10 depicts a portion of the overlay of substrate and product mixture. The spectrum of the substrate (methyl (Z) -hexadeca-11-enoate, 11Z-C16: 1-COOH) is shown in black and the product ((Z) -hexadeca-11-enal, 11Z-C16: -CHO; 12% The spectrum of (Z) -hexadeca-11-enol, 11Z-C16: 1-CH2OH) is shown in purple. [0056] FIG. 5 shows the collection of various spectra for Vitride reduction. FIG. 10A: Overlay of authentic standards [(Z) -hexadeca-11-enal and (Z) -hexadeca-11-enol] with the resulting mixture obtained in the Vitride reduction reaction. FIG. 10B: Observation of the presence of additional peaks in the spectrum. This was not seen in the spectra after one month when no derivatizing agent was used instead of BSTFA. FIG. 10C: Fragmentation pattern of authentic standard (Z) -hexadeca-11-enol. FIG. 10D: Fragmentation pattern of product peak at 4.1 minutes. FIG. 10E: Fragmentation pattern of authentic standard (Z) -hexadeca-11-enal. FIG. 10F: Fragmentation pattern of product peak at 5.75 minutes. [0057] FIG. 5 shows an overlay of the resulting mixture obtained by Vitride reduction. The main product (Z) -hexadeca-11-enal (5.7 minutes) is lost over time while the mixture is concentrated at 40 ° C. under vacuum. After 30 minutes relatively large amounts are seen, but after 150 minutes only about 25%. This also indicates that some of the product has been lost in vacuum for the first 30 minutes. This step can be further considered for purification purposes. [0058] Codon Optimization FIG. 7 shows that only zea desaturase variants produce detectable Z11-hexadecenoic acid in the SPV 140 screen. Control: pPV101 integrate SPV140, T.I. ni natural = natural codon usage T. ni Z11 desaturase (pPV 195), T. coli. ni HS opt = Homo sapiens codon optimization T. ni Z11 desaturase (pPV 196), T. coli. ni HS opt Yl leader = Homo sapiens codon optimization and exchanged Y. T. lipolytica OLE 1 leader sequence. ni Z11 desaturase (pPV 197); zea natural = natural codon usage H. zea Z11 desaturase (pPV198); zea HS opt = Homo sapiens codon optimization zea Z11 desaturase (pPV199); zea HS opt Yl leader = Homo sapiens codon optimization and exchanged Y. H. lipolytica OLE 1 leader sequence. zea Z11 desaturase (pPV200), A. transitella natural = natural codon usage A. transitella Z11 desaturase (pPV201). All data are the average of 3 biological replicates. Error bars indicate standard deviations. [0059] Codon Optimization FIG. 5 shows that only zea desaturase variants produce detectable Z11-hexadecenoic acid in the SPV 300 screen. The labels indicate the parent strain and the plasmid of the desaturase expression cassette. pPV101 = hrGFP control, pPV198 = natural codon usage zea Z11 desaturase, pPV 199 = Homo sapiens codon optimization zea Z11 desaturase, pPV200 = Homo sapiens codon optimization and exchange H. lipolytica OLE 1 leader sequence. zea Z11 desaturase, pPV 201 = natural codon usage transitella Z11 desaturase. [0060] FIG. 16 shows final cell densities for the desaturase screen on SPV140 and SPV300 background. The SPV300 strain with the inserted desaturase cassette grew to higher cell density. [0061] H. sapiens codon optimization FIG. 5 shows the individual isolated Z11-hexadecenoic acid titers of SPV140 and SPV300 strains expressing zea Z11 desaturase. [0062] Figure 12 shows Z11-14 acid (supplied with methyl myristate-14ME) and Z11-16 acid (supplied with methyl palmitate-16ME) titers of characterized Δ11 desaturases. SPV300 = Desaturase library insertion parent. SPV 298 = Prototrophic parent of SPV 300, negative control. SPV 459 = SPV 300 (Helicoverpa zea, SEQ ID NO: 1) with the currently optimal desaturase, positive control. Desaturase in DST 006 is expressed in SPV 459. Genetically equivalent to zea desaturase and serves as an internal library control. [0063] The C14 and C18 production profiles of SPV 298 (negative control, parent strain) and SPV 459 (strain SPV 298 with H. zea desaturase, SEQ ID NO: 1) supplied with methyl palmitate (16 ME) or methyl myristate (14 ME) are shown FIG. [0064] FIG. 24 depicts a downstream process (DSP) overview. Shows individual unit operations of the DSP process. [0065] Figure 12 shows a GC-FID trace of Zll-hexadecenol starting material. [0066] Figure 5 shows a GC-FID trace of the oxidation product of TEMPO bleach oxidation from Z11-hexadecenol to Z11-hexadecenal. [0067] Fig. 1 shows a phylogenetic tree of Z11-16 acid-producing strains. Strain lines for all strains are described in Example 6. The genotypes of SPV1199 and SPV1200 are identical but derived from different parent strains. [0068] FIG. 22 shows growth rate and production of Z11-16 acid in high glucose medium at both 28 ° C. and 32 ° C. FIG. 22A: Initial growth rate during the first 7 hours before the first substrate addition. Calculated from online cell density measurements. FIG. 22B: Maximum specific productivity (7-23 hours). Cell density is obtained from independent measurements using a plate reader. FIG. 22C: 48 hours (maximum observed) Z11-16 acid titer. The numbers inserted indicate the number of independent measurements. [0069] FIG. 7 shows initial growth rates of control and GUT2 deficient strains in high glucose medium without glycerol. The initial growth rate was smaller for all four strains tested with media lacking 5 g / L glycerol. [0070] Figure 7 shows the greatest increase in specific productivity in high glucose media with desaturase copy number. The average specific productivity of each strain in the SPV 963, SPV 968, and SPV 969 lines is represented by points. The single copy point is the average specific productivity of SPV 458. The exact copy number is unknown because of the presence of flanking repeat sequences in SPV 459. The average specific productivity of SPV 459 is shown in dotted lines. All three H. Strains SPV1056, SPV1 199, and SPV1200 with the zea Z11 desaturase copy and containing FAT1 deletion are outlined in red. Cultivation of these strains at 32 ° C. showed a decrease in specific productivity. FIG. 24A: Average specific productivity at 28 ° C. as a function of desaturase copy number. FIG. 24B: Average specific productivity at 32 ° C. as a function of desaturase copy number. [0071] FIG. 25 shows the correlation of performance index of Z11-16 acid in high glucose medium. FIG. 25A: The 48 hour titer of Z11-16 acid is moderately correlated with the maximum specific productivity observed. The correlation is improved at 32 ° C. FIG. 25B: 48 hour titer and selectivity of Z11-16 acid are also moderately correlated. FIG. 25C: 23 h and 48 h selectivity of Z11-16 acid is weakly correlated at 32 ° C. FIG. 25D: The 48 hour titer of Z11-16 acid does not correlate well with the putative lipid fraction. FIG. 25E: The 23 hour titer and selectivity of Z11-16 acid is strongly clustered. FIG. 25F: Estimates of total lipid fraction do not correlate with observed maximum specific productivity. [0072] FIG. 7 shows the selectivity and estimated lipid fraction of Z11-16 acid at both 28 ° C. and 32 ° C. in high glucose medium. FIG. 26A: Selectivity of Z11-16 acid at 48 hours. FIG. 26B: The lipid fraction (total fatty acid / dry cell weight) was estimated using the measured fatty acid concentration by multiplying the measured OD600 cell density by the factor 0.78 g DCW / L / OD600 previously observed. The numbers inserted indicate the number of independent measurements. TFA = total fatty acids, DCW = dry cell weight. [0073] FIG. 7 shows initial growth rates of HSD062 comparing high glucose and high glycerol media. Initial growth rates were similar in both media. [0074] FIG. 28 shows strain-specific responses to high glycerol medium and high glucose medium at 28 ° C. FIG. 28A: Specific productivity of Z11-16 acid for 7-23 hours. FIG. 28B: Specific productivity of Z11-16 acid for 7-41.5 hours. FIG. 28C: Z11-16 acid titer at 41.5 hours. FIG. 28D: Selectivity of Z11-16 acid at 41.5 hours. FIG. 28E: Estimated lipid fraction (total fatty acid / dry cell weight) at 41.5 hours. The lipid fraction was estimated using the measured fatty acid concentration by multiplying the measured OD600 cell density by the factor 0.78 g DCW / L / OD600 previously observed. [0075] Figure 5 shows fatty acid titers from HSD062. Upper row: Titers in high glucose medium (FERM1). Bottom row: Titers in high glycerol medium (FERM 3). [0076] FIG. 1 shows total fatty acid (TFA) titers from HSD062. Upper row: TFA titer in high glucose medium (FERM1). Bottom row: TFA titer in high glycerol medium (FERM 3). [0077] FIG. 31 is a diagram showing the performance parameters of Z11-16 acid B _d that contain data from all wells sample was taken. Several replicate cultures were incubated under hypoxic conditions if substrate / biofilm was formed under the plate seal of individual wells. Affected wells were identified by tracking dissolved oxygen concentration online. In addition, cell density and fatty acid titer were consistently lower in these wells. Here data is presented for all wells across all experiments. The numbers inserted indicate the number of independent measurements. FIG. 31A: Maximum specific productivity (7-23 hours). Cell density is obtained from independent measurements using a plate reader. FIG. 31B: 48 hours (maximum observed) Z11-16 acid titer. FIG. 31C: Selectivity of Z11-16 acid for 48 hours. FIG. 31D: The lipid fraction (total fatty acid / dry cell weight) was estimated by multiplying the measured OD600 cell density by the previously observed factor 0.78 g DCW / L / OD600, using the measured fatty acid concentration. FIG. 31E: Example of on-line dissolved oxygen measurement showing hypoxic conditions of column 3 wells of HSD043. All wells were started under the same conditions and inoculated with the same strain. The seal was changed at 23 hours and 31 hours. Wells D03, E03 and F03 were hypoxic with different durations during the period after the addition of substrate (7 hours) and the first sampling (23 hours). [0078] Figure 7 shows examples of acyl CoA intermediates generated by selective beta oxidation controlled by acyl CoA oxidase activity.

Array
[0079] The sequence listing of SEQ ID NO: 1 to SEQ ID NO: 47 is part of the present application and is incorporated herein by reference. A sequence listing is provided at the end of this document.

Detailed description definition
[0080] The following definitions and abbreviations will be used for the description of the present disclosure.

[0081] As used in the specification and the appended claims, the singular forms "a", "an" and "the" mean that It includes multiple references unless explicitly stated in the method of Thus, for example, reference to "a pheromone" includes a plurality of such pheromone, reference to "the microorganism" includes reference to one or more microorganisms, etc. .

[0082] As used herein, the terms "comprises", "comprising", "includes", "including", "has", " "Having", "contains", "containing" or any other variations are intended to cover non-exclusive inclusions. The composition, mixture, process, method, item or apparatus comprising the list of elements is not necessarily limited to just those elements, but may or may not be specifically listed as such composition, mixture , Processes, methods, items or other elements not inherent in the apparatus. Further, unless expressly stated to the contrary, "or" is inclusive (or) and not exclusive (or). .

[0083] The terms "about" and "around" as used herein to modify a numerical value indicate a short distance that surrounds its explicit value. Assuming that "X" is a value, "about X" or "approximately X" has a value of 0.9X to 1.1X, or in some embodiments, a value of 0.95X to 1.05X. Indicates Any reference to "about X" or "about X" specifically refers to at least X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1 The values of .03X, 1.04X, and 1.05X are shown. Thus, "about X" and "approximately X" are intended to teach and provide specification support for claim limitations, such as, for example, "0. 98X."

[0084] As used herein, the terms "microbial", "microbial organism", and "microorganism" are archaebacteria, bacteria or eukaryotes. The latter kingdom includes any organisms that exist as tiny cells contained within the kingdom, including yeast and filamentous fungi, protozoa, algae, or higher protozoa. Thus, the term is intended to encompass prokaryotic or eukaryotic cells or organisms of microscopic size, including all species of bacteria, archaebacteria and eubacteria and eukaryotic microorganisms such as yeast and fungi. It includes cell cultures of any species that can be cultured for the production of chemicals.

[0085] As described herein, in some embodiments, the recombinant microorganism is a prokaryotic microorganism. In some embodiments, the eukaryotic microbe is a bacterium. "Bacteria" or "eubacteria" is a prokaryotic kingdom. The bacteria are at least 11 different groups such as: (1) Gram-positive (gram +) bacteria, among which two major subdivisions: (1) High G + C (Actinomycetes, Mycobacteria, Micrococcus, others) ( 2) Low G + C groups (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas) are present; (3) Cyanobacteria, such as oxygenic phototrophs; (4) spirochetes and related species; (5) planktons; (6) Bacteroides, flavobacteria; (7) chlamydi. (8) green sulfur bacteria; (9) green non-sulfur bacteria (also anaerobic photosynthetic organisms); (10) radiation resistant micrococci and related species; (11) Termotoga and Thermosipho thermophiles including.

[0086] "Gram-negative bacteria" include cocci, nonenteric rods, and enteric rods. The genus of gram-negative bacteria is, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Bacteriobacteria, Bacillus subtilis, and , Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0087] "Gram-positive bacteria" include cocci, nonsporulating rods, and sporulating rods. The genus of gram positive bacteria includes, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0088] The terms "recombinant microorganism" and "recombinant host cell" are used interchangeably herein to express or overexpress an endogenous enzyme in a vector, such as an enzyme contained in the integration construct or the like. A microorganism that has been genetically modified to express an enzyme, or has altered expression of an endogenous gene. "Modification" means the expression of a gene encoding one or more polypeptides or polypeptide subunits, or the level of an RNA molecule or equivalent RNA molecule, or of one or more polypeptides or polypeptide subunits. It is meant that the activity is upregulated or downregulated such that expression, levels, or activity is greater or less than that observed in the absence of a change. For example, the term "alter" may mean "inhibit," but the use of the word "alter" is not limited to this definition. The terms "recombinant microorganism" and "recombinant host cell" are understood to mean not only the specific recombinant microorganism but also the progeny or potential progeny of such a microorganism. Such offspring may in fact not be identical to the parent cell, as certain modifications may occur in the next generation due to mutations or environmental influences, but are still used herein Are included within the term.

[0089] The term "expression" with respect to a gene sequence refers to transcription of the gene and, optionally, translation of the resulting mRNA transcript into a protein. Thus, as will be clear from context, protein expression results from transcription and translation of open reading frame sequences. The level of expression of the desired product in the host cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of desired product encoded by the selected sequence. For example, mRNA transcribed from selected sequences can be quantified by qRT-PCR or by Northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). ). The protein encoded by the selected sequence may be assayed by various methods, for example by ELISA, by assaying for the biological activity of the protein or using such an antibody which recognizes and binds to the protein. It is possible to quantify by using an assay such as an irrelevant Western blotting or radioimmunoassay. Sambrook et al., 1989, supra.

[0090] The term "polynucleotide" is used interchangeably herein with the term "nucleic acid" and is single- or double-stranded, sense or antisense deoxyribonucleic acid (DNA) of any length, and Correspondingly, two or more nucleotides, nucleosides or analogs thereof, including but not limited to single-stranded or double-stranded DNA of any length, including siRNA, including but not limited to sense or antisense ribonucleic acid (RNA) It is an organic polymer composed of monomers. The term "nucleotide" refers to any of several compounds consisting of a ribose or deoxyribose sugar linked to a purine or pyrimidine base and to a phosphate group, which are the basic structural units of nucleic acids. The term "nucleoside" refers to a compound (as guanosine or adenosine) which consists of a purine or pyrimidine base combined with deoxyribose or ribose, in particular in a nucleic acid. The terms "nucleotide analogue" and "nucleoside analogue" refer to nucleotides or nucleosides in which one or more individual atoms are replaced by different atoms or by different functional groups, respectively. Thus, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. Polynucleotides of three or more nucleotides are also referred to as nucleotide oligomers or oligonucleotides.

[0091] It is understood that the polynucleotides described herein include "genes" and the nucleic acid molecules described herein include "vectors" or "plasmids." Thus, the term "gene", also referred to as "structural gene", refers to a polynucleotide encoding a particular sequence of amino acids that makes up all or part of one or more proteins or enzymes, eg, a gene May comprise regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine the conditions under which is expressed. The transcribed region of the gene can include introns, untranslated regions including 5'-untranslated regions (UTRs) and 3'-UTRs, as well as coding sequences.

[0092] The term "enzyme" as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which substance is generally or entirely It is possible to include enzymes that are comprised in part of the polypeptide (s) but that are comprised of different molecules, including polynucleotides.

[0093] As used herein, the term "non-naturally occurring" as used in the context of the disclosed microorganism, refers to a microorganism comprising a wild-type strain of the referenced species. It is intended to mean having at least one genetic mutation not normally found in naturally occurring strains of the species being considered. Genetic mutations include, for example, modifications that introduce expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and / or other functional disruptions of microbial genetic material. Such modifications include, for example, the coding region of the heterologous, homologous, or both heterologous and homologous polypeptides of the referenced species and functional fragments thereof. Additional modifications include, for example, non-coding regulatory regions whose expression alters the expression of the gene or operon. Exemplary non-naturally occurring microorganisms include recombinant microorganisms engineered for the production of unsaturated lipid moieties described herein.

[0094] In contrast, the term "naturally occurring" as used in connection with the organisms of the present disclosure, includes genetic engineering for the recombinant production of one or more unsaturated lipid moieties. It is intended to mean an organism that has not been engineered to include a biosynthetic pathway that has been engineered.

[0095] The term "exogenous" as used herein in relation to various molecules, such as polynucleotides, polypeptides, enzymes etc. refers to the naturally occurring yeast, bacteria, organism, microorganism Or molecules that are not normally or naturally found in cells and / or are not produced by them.

[0096] On the other hand, the terms "endogenous" or "native" as used herein in relation to various molecules, such as polynucleotides, polypeptides, enzymes etc. A molecule normally or naturally found in and / or produced by a given yeast, bacterium, organism, microorganism or cell.

[0097] As used herein in the context of a modified host cell, the term "heterologous" refers to the following: (a) the molecule (s) are foreign ("exogenous") to the host cell Some (i.e. not found naturally in the host cell); (b) the molecule (s) are found naturally in a given host microorganism or host cell (i.e. "is endogenous"), It is produced in non-naturally occurring locations or in non-naturally occurring quantities in cells; and / or (c) the molecule (s) differ from the nucleotide or amino acid sequence (s) in which the nucleotide or amino acid sequence is endogenous, Molecules which differ in nucleotide or amino acid sequence from endogenous nucleotides or amino acids which are found endogenously are only available in non-naturally occurring (eg, greater than naturally found) amounts in the cell. Refers to various molecules, such as polynucleotides, polypeptides, enzymes, etc., of which at least one of them is produced at a non-natural (eg, greater than naturally found) activity level in cells. .

[0098] The term "homologue" as used herein with respect to a first family or species native enzyme or gene refers to a second family that corresponds to the first family or species native enzyme or gene. Or a different enzyme or gene of a second family or species that is determined by functional, structural or genomic analysis to be a species enzyme or gene. Homologs almost always have functional, structural or genomic similarity. Techniques are known in which homologs of enzymes or genes can be easily cloned using gene probes and PCR. Identifying cloned sequences as homologs can be confirmed using functional assays and / or by genomic mapping of genes.

[0099] A protein is "homologous" or "homologous" to a second protein if the amino acid sequence encoded by the gene has an amino acid sequence similar to that of the second gene. Instead, the proteins are homologous to the second protein if the two proteins have "similar" amino acid sequences. Thus, the term "homologous protein" is intended to mean that the two proteins have similar amino acid sequences. In certain instances, homology between two proteins is indicative of their common ancestor linked by evolution.

[00100] The term "fatty acid" as used herein is a compound of structure R-COOH, wherein R is C ₆ to C ₂₄ saturated, unsaturated, linear, branched or cyclic hydrocarbon There is a carboxyl group at position 1. In certain embodiments, R is a C ₆ to C ₂₄ saturated or unsaturated linear hydrocarbon and the carboxyl group is at the 1 position.

[00101] The term "fatty alcohol" as used herein refers to a fatty alcohol having the formula R-OH, R is C ₂₄ saturated from C _6, unsaturated, linear, branched or It is a cyclic hydrocarbon. In certain embodiments, R is a C ₆ to C ₂₄ saturated or unsaturated linear hydrocarbon.

[00102] The term "fatty acyl CoA" refers to a compound having the structure R- (CO) -S-R ₁ , R ₁ is coenzyme A, and the term "fatty acyl ACP" refers to structure R It is a compound which has-(CO) -S-R ₁ and R ₁ is an acyl carrier protein ACP.

[00103] The term "fatty acid derivative" as used herein refers to any organic molecular entity derived from a fatty acid. Fatty acid derivatives include, but are not limited to, saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, branched fatty acids, triacylglycerols (triglycerides) and alkyl esters of fatty acids.

[00104] The term "semibiosynthesis" as used herein is to synthesize a product by combining biological and non-biological means. In one embodiment, the biological means comprises obtaining one or more compounds or molecules from one or more natural sources, such as naturally occurring organisms. In some embodiments, one or more naturally occurring organisms can be plants, bacteria, algae, yeast, and / or cell cultures. In another embodiment, the biological means comprises obtaining one or more compounds or molecules from one or more recombinant microorganisms engineered to produce the compounds or molecules . These compounds or molecules can be further modified to the final product by non-biological means such as chemical synthesis. Chemical synthesis is the preparation of compounds by performing various chemical reactions using starting materials and changing their molecular structure by reaction with other chemicals. Starting materials for organic synthesis can be simple compounds removed from petroleum and natural gas or more complex chemicals isolated in large quantities from plant and animal sources. In certain embodiments, the semibiosynthesis of one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is one or more naturally occurring organisms. Obtaining one or more unsaturated lipid moieties from or one of one or more recombinant microorganisms engineered to include a biosynthetic pathway for the production of one or more unsaturated lipid moieties Producing one or more unsaturated lipid moieties; chemically converting one or more unsaturated lipid moieties to one or more free fatty acids (FFA) and / or one or more fatty acid methyl esters (FAAE) Converting, and reducing one or more FFAs and / or one or more FAAEs, Include producing a plurality of fatty alcohols and / or one or more fatty aldehydes.

[00105] The term "unsaturated lipid moiety" as used herein refers to a lipid or lipid derivative having one or more double bonds. Lipids are biological molecules that are insoluble in aqueous solution and soluble in organic solvents. Unsaturated lipid moieties can include, but are not limited to, monounsaturated fatty acids, polyunsaturated fatty acids, branched unsaturated fatty acids and unsaturated triacylglycerols (unsaturated triglycerides). Unsaturated lipid moieties can include fats, sterols, waxes, oils, fat-soluble vitamins, phospholipids, sphingolipids, plasmalogens, eicosanoids, terpenes, and soaps and surfactants.

[00106] The term "non-biological conversion" as used herein refers to the conversion of one or more substrates into one or more products using chemical means. In one embodiment, one or more unsaturated lipid moieties are chemically converted to one or more FFAs and / or one or more FAAEs. In another embodiment, one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs are one or more unsaturated lipid moieties, one or more FFAs and And / or one or more stoichiometries of one or more FAAEs such as sodium bis (2-methoxyethoxy) aluminum hydride Vitride (Red-Al, Vitride, SMEAH) and / or diisobutylaluminum hydride (DIBAL) Contact with a reducing agent reduces it to one or more fatty alcohols and / or one or more fatty aldehydes. Additionally, using a bulky cyclic nitrogen Lewis base, it is possible to modify the reducing agent to provide selectivity of the reduction step for the production of one or more fatty aldehydes. In another embodiment, reducing one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs to one or more fatty alcohols comprises one or more of: Contacting the unsaturated lipid moiety, one or more FFAs and / or one or more FAAEs with one or more transition metal catalysts, such as one or more Group VIII catalysts. In another embodiment, the fatty alcohol produced is further oxidized to a fatty aldehyde by an oxidation process. The oxidation step can be one or more partial oxidation methods such as NaOCI / TEMPO. In another embodiment, the oxidation step comprises copper catalyzed aerobic oxidation.

Introduction
[00107] The present disclosure addresses the need for new technologies for cost effective production of valuable products from low cost feedstocks. Specifically, we obtain or produce one or more unsaturated lipid moieties from a biological source, one or more fatty alcohols and / or one or more unsaturated lipid moieties. Produces one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties by combining with conversion by one or more fatty aldehydes into non-biological means We have addressed this need by developing methods to One or more fatty alcohols and / or one or more fatty aldehydes may be useful as insect pheromone, flavors, flavors and / or polymer intermediates. Thus, aspects of the present disclosure produce unsaturated lipid moieties by recombinant microorganisms obtained from naturally occurring organisms or engineered to produce unsaturated lipid moieties from low cost feedstocks. One or more fatty alcohols and / or ones useful as various valuable products including, but not limited to, insect pheromone, perfumes, flavors and polymeric intermediates. Or based on our findings that can be combined with conventional synthetic methodologies to produce multiple fatty aldehydes.

[00108] The application further relates to recombinant microorganisms that are useful for biosynthesis of unsaturated lipid moieties, as well as compositions comprising one or more fatty alcohols and / or one or more fatty aldehydes. Thus, using various embodiments of the present disclosure, it is possible to synthesize various insect pheromone selected from fatty alcohols, aldehydes, and acetic acid. In addition, the embodiments described herein can also be used in the synthesis of perfumes, flavors and polymer intermediates.

Pheromones
[00109] As mentioned above, embodiments of the present disclosure provide semi-biosynthesis of one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties. One or more fatty alcohols and / or one or more fatty aldehydes may be useful as insect pheromone. Pheromones are volatile chemicals that are secreted by certain insects for the purpose of chemical communication within species. That is, pheromone is a secreted or excreted chemical agent that elicits a social response in members of the same species. Among them are alarm pheromone, food trail pheromone, sex pheromone, aggregating pheromone, epidetictic pheromones, inspiring pheromone, primer pheromone, and territorial pheromone, which affect behavior or physiology.

[00110] Non-limiting examples of insect pheromone that can be synthesized using the recombinant microorganisms and methods disclosed herein include the linear alcohols, aldehydes, and acetic acid listed in Table 1 including.

[00111] In some aspects, one or more fatty alcohols and / or one or more fatty aldehydes produced by the methods taught in the present disclosure may be useful as insect pheromone. Exemplary pheromone that modulate insect behavior are listed in Table 2. However, the methods and microorganisms described herein is not limited to the synthesis of C ₆ -C ₂₀ pheromones are listed in Table 1 and Table 2. Rather, the disclosed methods and microorganisms can also be utilized for the synthesis of various fatty alcohols, fatty aldehydes, and fatty acetic acids, including perfumes, flavors and polymeric intermediates.

[00112] Most pheromones contain a hydrocarbon backbone in which terminal hydrogens are substituted with functional groups (Ryan MF (2002). Insect Chemoreception. Fundamental and Applied. Kluwer Academic Publishers). Table 2b shows some common functional groups together with the formula, prefixes and suffixes. The presence of one or more double bonds produced by the elimination of hydrogen from the adjacent carbon determines the degree of unsaturation of the molecule, from-ane (without multiple bonds) to-ene Change the name of the hydrocarbon up to). The presence of two and three double bonds is indicated by ending the name with -diene and -triene respectively. The position of each double bond is represented by a number corresponding to the position of the carbon where the double bond begins, and each carbon is numbered from the carbon attached to the functional group. The carbon attached to the functional group is designated as -1-. Pheromones may have hydrocarbon chain lengths that count with 10 (deca-), 12 (dodeca-), 14 (tetradeca-), 16 (hexadeca-), or 18 (octadeca-) carbon lengths but are limited to these I will not. The presence of a double bond has another effect. The double bond represents a geometric isomer, which is a different molecule, preventing rotation of the molecule by immobilizing the molecule in one of two possible configurations. They are named E (German Entgegen, from the opposite) or Z (Zusammen, together), the carbon chains being connected on the opposite (trans) or the same (cis) side of the double bond respectively .

[00113] Pheromones described herein can be referred to using the IUPAC nomenclature or various abbreviations or variations known to those skilled in the art. For example, (11Z) -hexadecen-1-al can also be written as Z-11-hexadecen-1-al, Z-11-hexadecenal, Z-11-16: Ald, or Zxy: Ald And x represents the position of the double bond and y represents the number of carbon atoms in the hydrocarbon backbone. The abbreviations used herein to identify functional groups on hydrocarbon backbones and known to those skilled in the art are "Ald" aldehyde, "OH" alcohol, and "Ac" acetyl, including. Moreover, the number of carbons in the chain can be indicated using numbers rather than using written names. Thus, as used herein, unsaturated carbon chains composed of 16 carbons can be written as hexadecene or 16.

[00114] Similar abbreviations and word sources are used herein to describe pheromone precursors. For example, the fatty acyl-CoA precursor of (11Z) -hexadecene-1-al can be identified as (11Z) -hexadecenyl-CoA or Z-11-16: acyl-CoA. The abbreviation representing a carboxylic acid functional group is "COOH". Fatty acids are carboxylic acids with long hydrocarbon chains. In the IUPAC nomenclature, the carboxyl carbon is C-1. The generic nomenclature refers to C-1 to alpha, beta, gamma, delta, epsilon, etc., and the carbon most distant from the carboxyl is omega. Fatty acids: all # carbon: # double bonds, an abbreviation describing the double bond position, the position of the double bond is indicated by Δ ⁿ , and n is the number of each pair in which a double bond is present Indicates the less carbon. For example, 18: 3Δ8t, Δ10t, Δ12c refer to fatty acids that are 18 carbons long with three double bonds at positions C8, C10 and C12 of the hydrocarbon chain. The double bond at positions C8 and C10 is in trans (t) configuration and the double bond at position C12 is in cis (c) configuration.

[00115] In one aspect, a method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is one or more naturally occurring organisms. Comprising obtaining one or more unsaturated lipid moieties from

Naturally occurring organisms as a source of unsaturated lipid moieties
[00116] In one embodiment, the one or more naturally occurring organisms are one or more insects. In another embodiment, the one or more insects include Coleoptera, Diptera, Hymenoptera and Lepidoptera.

[00117] In another embodiment, the one or more naturally occurring organisms are not insects.

[00118] In some embodiments, the one or more naturally occurring organisms comprise one or more plants. In some embodiments, the plant comprises soybean, canola, cotton, sunflower, peanuts, coconut, coconut, olive, sesame and / or linseed.

[00119] In one embodiment, soybean is a member of the genus Glycine. In another embodiment, the soybean is Glycine max. The main unsaturated fatty acids in soybean oil triglycerides are polyunsaturated alpha linolenic acid (C18: 3, about 7% to about 10%), and linoleic acid (C18: 2, about 51%); and monounsaturated It is oleic acid (C18: 1, about 23%). Soy oil also contains saturated fatty acid stearic acid (C18: 0, about 4%) and palmitic acid (C16: 0, about 10%). Changes in fatty acid profiles can occur due to genetics and / or climate.

[00120] In one embodiment, canola is a member of the family Brassicaceae. In another embodiment, the canola is a member of the genus Brassica. In certain embodiments, the canola is selected from the group consisting of Brassica napus, Brassica rapa, Brassica oleracea, and Brassica juncea. Canola oil may have about 61% oleic acid, about 21% linoleic acid, about 9% to about 11% alpha linolenic acid, about 4% palmitic acid and about 2% stearic acid, the rest The proportion of is another fatty acid. Changes in fatty acid profiles can occur due to genetics and / or climate. Commercial food grade canola oil is grown to a content of erucic acid (C22: 1) less than 2%.

[00121] In one embodiment, the cotton is a member of the genus Gossypium. In another embodiment, the cotton is selected from the group consisting of Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum and Gossypium herbaceum. Cotton has oil-bearing grains surrounded by a hard crust. Cottonseed oil may have about 52% linoleic acid, about 17% oleic acid, and about 24% palmitic acid, with the remaining proportion being other fatty acids. Changes in fatty acid profiles can occur due to genetics and / or climate.

[00122] In one embodiment, the sunflower is a member of the genus Helianthus. In another embodiment, the sunflower is Helianthus agrestis, Helianthus ambiguous, Helianthus angustifolius, Helianthus annuus, Helianthus anomalus, Helianthus argophylus, Helianthus alirubutus, Helihiglih, as a cirrhitis , Helianthus coloradensis, elianthus cusickii, Helianthus debilis, Helianthus decapetalus, Helianthus deserticola, Helianthus diffuses, Helianthus dissectifolius, Helianthus divaricatus, Helianthus × divariserratus, Helianthus × doronicoides, Helianthus eggertii, Helianthus exilis, Helianthus floridanus, Helianthus giganteus, Helianthus glaucophyllus, Helianthus × glaucus, Helianthu gracilentus, Helianthus grosseserratus, Helianthus heterophyllus, Helianthus hirsutus, Helianthus × intermedius, Helianthus laciniatus, Helianthus × laetiflorus, Helianthus laevigatus, Helianthus lenticularis, Helianthus longifolius, Helianthus × luxurians, Helianthus maximiliani, Helianthus membranifolius, Helianthus mollis, Helianthus multiflorus, Helian thus navarri, Helianthus neglectus, Helianthus niveus, Helianthus nuttallii, Helianthus occidentalis, Helianthus × orgyaloides, Helianthus paradoxus, Helianthus pauciflorus, Helianthus petiolaris, Helianthus porteri, Helianthus praecox, Helianthus praetermissus, Helianthus pumilus, Helianthus radula, Helianthus resinosus, Helianthus salicifolius, Heliant Helianthus, Heianthus sarmentosus, Heianthus sarmentosus, Heianthus sarmentosus, Heianthus, Heianthus sarmentosus, Heianthus scalus, Heianthus scalus, Heianthus scavirimus, Heianthus scavirimus, Heianthus scavirimus, Heli The sunflower oil may have about 5% palmitic acid, about 6% stearic acid, about 30% oleic acid and about 59% linoleic acid. However, several types of sunflower oil have been produced, such as high linoleic acid, high oleic acid and intermediate oleic acid. Intermediate oleic acid sunflower oil typically has at least 69% oleic acid. High oleic acid sunflower oil has at least 82% oleic acid. Changes in unsaturated fatty acid profiles are strongly influenced by both genetics and climate.

[00123] Peanuts are also known as peanuts. In one embodiment, the peanut is a member of the genus Arachis. In another embodiment, the peanut is Arachis hypogaea. Peanut oil may have about 46% to about 47% oleic acid, about 33% linoleic acid and about 10% palmitic acid. Peanut oil may also have stearic acid, arachidic acid (eicosanoic acid, C20: 0), behenic acid (docosanoic acid, C22: 0), lignoceric acid (tetracosanoic acid, C24: 0) and other fatty acids. Changes in fatty acid profiles can occur due to genetics and / or climate.

[00124] In one embodiment, the palm is a member of the Arecaceae family. In another embodiment, the palm is a member of the genus Elaeis. In a particular embodiment, the palm is selected from the group consisting of Elaeis guineensis (Africa oil palm) and Elaeis oleifera (American oil palm). Palm oil is derived from the mesocarp or reddish flesh of the fruit. Coconut oil is about 43.5% palmitic acid, about 36.6% oleic acid, about 9.1% linoleic acid, about 4.3% stearic acid, about 1% myristic acid (C14: 0) And may have about 5.5% other fatty acids. The seeds, or palm kernel, are also rich in oil. Palm kernel oil is more saturated than coconut oil and is about 48% lauric acid (C12: 0), about 16% myristic acid, about 15% oleic acid, about 8% palmitic acid, about 3% It may have capric acid (C10: 0), about 3% caprylic acid (C8: 0), about 2% to about 3% stearic acid and about 2% linoleic acid. The remaining proportions include other fatty acids. Changes in fatty acid profiles can occur due to genetics and / or climate.

[00125] In one embodiment, the coconut is a member of the Arecaceae family. In another embodiment, the coconut is a member of the genus Cocos. In one particular embodiment, the coconut is Cocos nucifera. Coconut oil has about 51% lauric acid, about 19% myristic acid, about 8% to about 9% palmitic acid, about 6% caprylic acid, about 6% capric acid and about 6% oleic acid Sometimes. The remaining proportions include other fatty acids. Changes in fatty acid profiles can occur due to genetics and / or climate.

[00126] In one embodiment, the olive is a member of the Oleaceae family. In another embodiment, the olive is a member of the genus Olea. In one particular embodiment, the olive is Olea europaea. Olive oil is mainly a mixed triglyceride ester of oleic acid (about 55% to about 83%) and palmitic acid (about 7.5% to about 20%) and other fatty acids (linoleic acid: about 3.5% to about 21) Stearic acid: about 0.5% to about 5%); alpha linolenic acid (about 0% to about 1.5%) and a slight amount of squalene (up to about 0.7%) and sterols (about 0.2%) And Phytosterols and Tocosterols). Composition varies with cultivar, area, altitude, harvest time, and extraction process.

[00127] In one embodiment, the sesame is a member of the genus Sesamum. In another embodiment, the sesame is Sesamum indicum. Sesame oil has about 41% linoleic acid, about 39% oleic acid, about 8% palmitic acid, about 5% stearic acid, and the balance may be composed of other fatty acids. Changes in fatty acid profiles can occur due to genetics and / or climate.

[00128] Flaxseed is also known as flax or hemp. In one embodiment, the linseed is a member of the Linaceae family. In another embodiment, the linseed is a member of the genus Linum. In certain embodiments, the linseed is Linum usitatissimum. Flaxseed oil, also known as flaxseed oil, is a colorless to yellowish oil obtained from dried dried seed of flax. Flaxseed oil is characterized by its abnormally high amount of alpha linolenic acid (omega-3 fatty acids), about 51.9% to about 55.2%. Flaxseed oil comprises saturated acid palmitic acid (about 7%) and stearic acid (about 3.4% to about 4.6%); monounsaturated oleic acid (about 18.5% to about 22.6%); It may also have saturated linoleic acid (about 14.2% to about 17%). Changes in fatty acid profiles can occur due to genetics and / or climate.

[00129] In some embodiments, the plant is from a family selected from the group consisting of pokeweed family and buckthorn family. The Magnolia family is a family of flowering plants that are predominantly distributed in the southern hemisphere. The family includes about 80 genera, about 1600 species. Together, Platanaceae and Nelumbonaceae make up the Proteales eye. Well known genera include Protea, Banksia, Embothrium, Grevillea, Hakea, Dryandra and Macadamia. Seeds such as New South Wales Warata (waratah) (Telopea speciosissima), King Protea (Protea cynaroides), and various species of Banksia, Grevillea, and Leucadendron are popular cut flowers, and the trees of Macadamia integrifolia are commercially widely grown. Being consumed. Australia and South Africa have the highest concentration of diversity. Rhamnaceae is a large family of flowering plants, most of which are trees, shrubs, and some are vine plants, and are commonly referred to as the euphorbiosis family. The family contains 50 to 60 genera, roughly 870-900 species. Rhamnaceae is distributed around the world, but subtropics and tropical regions are more common.

[00130] In a specific embodiment, the plant is Gevuina, Calendula, Limnanthes, Lunaria, Carum, Daucus, Daucus, Coriandrum, Macadamia, Myristica, Licania, Aleurites, Ricinus, Corylus. And species from the genus Kermadecia, Asclepias, Cardwellia, Grevillea, Orites, Ziziphus, Hicksbeachia, Hipkshaesia, Hippophae, Ephedra, Placospermum, Xylomelum and / or Simmondsia.

[00131] Calendula is a genus of about 15 to 20 species of annual and perennial herbaceous plants of the daisy family Asteraceae and is often known as marigold. This plant is native to southwest Asia, Western Europe, Makaronenesia, and the Mediterranean. C. The oil of officinalis is used as an anti-inflammatory agent, an antineoplastic agent, and a therapeutic agent for healing wounds. Calendula species are: Calendula arvensis, Calendula denticulate, Calendula eckerleinii, Calendula incana, Calendula lanzae, Calendula maritima, Calendula maroccana, Calendula meuselisi, Calendula palaestina, Calendula stellate, Ticlitis c

[00132] Limnanthes is a reference genus of the Limnantheaceae family and consists of annual herbaceous plants commonly known as meadowfoams. All seven species are native to the coast and adjacent areas (inland valleys, foothills and mountains) of western North America, where these species typically grow in wetland habitats such as spring puddles. Some species are unique to California. This genus is divided into two subgenuses: Limnanthes, which scaly curls during fruit ripening, and Inflexae, which scallops bends on ripening fruits. The subgenus Limnanthes includes Limnanthes bakeri, Limnanthes douglasii, Limnanthes macounii and Limnanthes vinculans. Section Inflexae includes Limnanthes alba, Limnanthes floccose and Limnanthes montana.

[00133] Lunaria is a flowering plant genus of Brassicaceae and is native to central and southern Europe. Lunaria contains four species and is the annual or biennial Lunaria annua (L. biennis), Lunaria elongata, the perennial Lunaria rediviva and the rare Balkan Lunaria telekiana.

[00134] Carum is a genus of about 20 flowering plants of the Apiaceae family, and is native to the warm world of the Old World. The most important species is caraway (C. carvi), whose seeds are widely used as cooking flavours. In the Flora of Mongolia, Carum L. There are two species (C. carve L., C. buriaticum Turcz.) Belonging to the genus.

[00135] Daucus is a worldwide genus of herbaceous plants of the Apiaceae family, the most well-known of which are cultivated carrots. There are about 25 species of Daucus of Umbelliferae Apiaceae. The species is Daucus aureus, Daucus azoricus, Daucus broteri, Daucus bicolor, Daucus carota, Daucus durieui, Daucus foliosus, Daucus glochidiatus, Daucus guttatus, Daucus involucatus suchus, as in the case of Daucus i And Daucus visnaga.

[00136] Coriandrum is a genus of herbs of the Apiaceae family, and contains the cultivated species Coriandrum sativum (Corianda) and the wild species Coriandrum tordylium. The leaves and seeds of Coriandrum sativum are used for cooking. The leaves are often called coriander leaves in North America.

[00137] Macadamia is a genus of four species of Australian native trees and is a component of the bollworm family. The four species are native to northeastern New South Wales and central and southeastern Queensland. The tree is commercially important in its fruit, macadamia nuts. Other names include Queensland nuts, bush nuts, marouchi nuts, bauple nuts, and Hawaii nuts. Macadamia species include Macadamia integrifolia, Macadamia jansenii, Macadamia ternifolia and Macadamia tetraphylla. This nut has a large amount of monounsaturated fat (59% of the total). Macadamia oil is highly valued for containing approximately 22% omega 7 palmitoleic acid, which makes it a botanical substitute for mink oil. Due to the high oxidative stability of this relatively high content of palmitoleic acid plus macadamia macadamia oil has become a desirable component of cosmetics.

[00138] Myristica is a genus of trees of the Myristicaceae family. About 150 species are distributed in Asia and the West Pacific. The most important commercial species is Myristica fragrans, a major source of perfume nutmeg and mace. The species of Myristica is M. acsmithii, M. agusanensis, M. alba, M. albertisii, M. amboinensis, M.. ampliata, M .; amplifolia, M .; amygdalina, M. anceps, M. andamanica, M .; apiculate, M. archboldiana, M .; arfakensis, M. argentea, M. aruensis, M. atrescens, M .; atrocorticata, M. attenuate, M. avis-paradisiacae, M. a. baeuerlenii, M .; bancana, M .; basilanica, M. batjanica, M. beccarii, M .; beddomei, M .; bivalvis, M .; bombycina, M. brachiate, M. brachypoda, M .; brassii, M. brevistipes, M .; buchneriana, M .; byssacea, M. cagayanensis, M. canariformis, M. Cantleyi, M .; carrii, M. castaneifolia, M. celebica, M. cerifera, M. ceylanica, M. chartacea, M. chrysophylla, M. cimicifera, M. cinerea, M. cinnamomea, M .; clemensii, M. coacta, M. colinridsdalei, M .; collettiana, M .; commersonii, M. concinna, M .; conspersa, M .; contorta, M. contracta, M. cookie, M. coriacea, M. cornutiflora, M .; corticata, M. corticosa, M. costata, M. costulata, M .; crassa, M. crassifolia, M. crassinervis, M .; crassipes, M .; cucullata, M .; cumingii, M. curtisii, M. cylindrocarpa, M. dactyloides, M. dardaini, M. dasycarpa, M. depressa, M. devogelii, M. diversifolia, M .; duplopunctata, M. duthiei, M. C. elegans, M. elliptica, M .; ensifolia, M .; eugeniifolia, M. euryocarpa, M. extensa, M .; fallax, M. faroensis, M. farquhariana, M .; fasciculate, M. fatua, M. filipes, M. finlaysoniana, M .; firmipes, M. fissiflora, M. fissurata, M .; flavovirens, M .; flocculosa, M. flosculosa, M. forbesii, M. fragrans, M .; frugifera, M. fugax, M. furfurascerts, M. fusca, M. fusiformis, M. Gamblei, M .; Garciniifolia, M .; geminate, M. gibbosa, M .; gigantean, M. gillespieana, M .; globose, M. gracilipes, M .; grandifolia, M .; grandis, M. griffithii, M .; guadalcanalensis, M. guatteriifolia, M .; guillauminiana, M .; hackenbergii, M .; hellwigii, M .; heritierifolia, M .; hollrungii, M .; hooglandii, M .; horsfieldia, M. hypargyraea, M .; hypoposticta, M. impressa, M. impressinervia, M. inaequalis, M. incredibilis, M .; iners, M. ingens, M .; inrate, M. inopinata, M. insipida, M. intermedia, M. inundata, M. inutilis, M. irya, M. iteophylla, M .; johnsii, M .; kajewskii, M. kalkmanii, M. kjellbergii, M. koordersii, M. korthalsii, M .; kunstleri, M .; kurzii, M .; laevifolia, M .; laevigata, M. laevis, M. lakilaki, M. lasiocarpa, M. laurella, M .; laurina, M .; laxiflora, M .; lemanniana, M. lenta, M .; lepidota, M .; leptophylla, M .; leucoxyla, M. litoralis, M .; longipes, M. longipetiolata, M. lowiana, M .; macgregori, M. macrantha, M .; macrocarpa, M. macrocarya, M. macrocoma, M. macrothyrsa, M. magnifica, M. maingayi, M. majuscule, M. malabarica, M. malayana, M .; mandaharan, M .; markgraviana, M. mascala, M .; maxima, M. mediovibex, M. mediterranea, M .; micrantha, M .; microcarpa, M. millepunctata, M. mindanaensis, M.. mindorensis, M.. miohu, M. mouchio, M. multinervia, M. Murtoni, M .; myrmecophila, M. nana, M. neglecta, M .; negrosensis, M. Nesophila, M., et al. niobue, M .; niohne, M .; nitida, M .; nivea, M. oblongifolia, M. olivacea, M .; orinocensis, M.. ornate, M. ovicarpa, M. pachycarpidia, M .; pachyphylla, M .; pachythyrsa, M .; palawanensis, M.. paludicola, M .; papillatifolia, M .; papuana, M .; papyracea, M .; parviflora, M. Pectinate, M .; pedicellata, M .; peltata, M .; pendulina, M .; perlaevis, M. petiolate, M .; philippensis, M. pilosella, M .; pilosigemma, M. pinnaeformis, M .; platysperma, M .; plumeriifolia, M. polyantha, M. polyspherula, M. pseudoargentea, M. psilocarpa, M. pubicarpa, M .; pulchra, M .; pumila, M .; pygmaea, M. quercicarpa, M .; racemose, M. radja, M. resinosa, M. retusa, M .; ridleyana, M .; ridleyi, M .; riedelii, M .; robusta, M. rosselensis, M. rubiginosa, M. rubrinervis, M .; rumphii, M .; sagotiana, M. salomonensis, M.. sangowoensis, M. sapida, M. sarcantha, M .; schlechteri, M. schleinitzii, M. schumanniana, M. scortechinii, M .; scripta, M. sericea, M. sesquipedalis, M. simiarum, M .; simulans, M. sinclairii, M .; smythiesii, M. sogeriensis, M. spanogheana, M .; sphaerosperma, M .; sphaerula, M .; spicata, M. sprucei, M. stenophylla, M .; suavis, M. subalulata, M. subglobosa, M. subtilis, M. succedanea, M .; succosa, M. sulcata, M. suluensis, M .; sumbavana, M. superba, M. tamrauensis, M .; teijsmannii, M. tenuivenia, M .; teysmanni, M .; Tingens, M .; tomentella, M. tomentosa, M. trianthera, M .; tristis, M .; tuberculate, M. tubiflora, M .; ultrabasica, M. umbellate, M .; umbrosa, M .; uncinata, M. undulatifolia, M .; urdanetensis, M. uviformis, M. valida, M. velutina, M .; verruculosa, M .; villosa, M. vinkeana, M .; vordermanni, M. wallaceana, M. wallichii, M. warburgii, M. wenzelii, M. womersleyi, M. wrayi, M. wyatt-smithii, M .; yunnanensis and M. It is possible to include zeylanica.

[00139] Licania is a genus of plants of the family Chrysobalanaceae. Several species are used as ornamental plants. Licania fruit is an important food for many animals and can be eaten by humans. The species of Licania are Licania arborea, Licania caldasiana, Licania chiriquiensis, Licania conferruminata, Licania fasciculate, Licania grandibracteata, Licania hedbergii, Licania kunthic, Licania longiculus ideicelli, which is a snail, as well as Licania longiculusi rigida, Licania salicifolia, Lica nia splendens, Licania tomentosa, Licania vasquezii and Licania velutina.

[00140] Aleurites is a small genus of dendritic flowering plants of Euphorbiaceae. Aleurites are native to China, the Indian subcontinent, Southeast Asia, Papuasia, and Queensland. Aleurites are reportedly ported to various islands (Pacific and Indian Ocean, plus Caribbean) and also to scattered locations in Africa, South America, and Florida. These hermaphrodite evergreen trees are perennial or semi-perennial. The fruits are quite large stone fruits, with fleshy outer peel and thin woody inner peel. The fruit changes shape according to the number of developed ovary. The fruits contain oleotoxic seeds. The oils have been used as kerosene, lubricants and as components of varnishes, paints and soaps. After removal of toxic substances, the oil can be used as a cooking oil. The species of Aleurites is A. cordatus, A. erraticus, A. fordii, A. japonicas, A. laccifer, A. montanus A. peltatus, A. saponarius, A. trispermus A. vernicifluus and A. Includes vernicius.

[00141] Ricinus communis is a castor bean or castor bean and is a species of flowering plants of the family Euphorbiaceae, Euphorbiaceae. Ricinus communis is the only species of a single genus, Ricinus, and a subfamily, Ricininae. The seed is castor bean, and despite its name it is not a real bean. It is native to the southeastern Mediterranean basin, East Africa and India, but is widely distributed throughout the tropics (otherwise it is widely grown as an ornamental plant). Castor oil is a source of castor oil, which has a wide range of uses. The seeds are rich in triglycerides and contain 40% to 60% of oil, mainly ricinoline. The seeds also contain ricin, a water soluble toxin, which is also present at lower concentrations throughout the plant.

[00142] Corylus (Hashabami) is a temperate northern hemisphere native deciduous tree and a large shrub genus. The genus is usually found in the birch, Betulaceae, but some botanists divide hazelrs into a different family, Corylaceae. The hazelnut is hazelnut. The fruits are 1 to 2.5 cm long and 1 to 2 cm in diameter and are surrounded by a sheath which partially and completely surrounds the nut. The species of Corylus is Corylus Americana, Corylus avellana, Corylus heterophylla, Corylus yunnanensis, Corylus colchica, Corylus cornuta, Corylus maxima, Corylus sieboldiana (C. mandshurica), Corylus chinensis, Corylus colurocytecilium, and is a member of the body, including including.

[00143] Kermadecia is a genus of flowering plants of the Phylumaceae family. This genus includes four species (K. elliptica, K. pronyensis, K. rotundifolia and K. sinuata) and all species are unique to New Caledonia. Kermadecia sinuata is a tree up to 30 m high, and its trunk is up to 1 m in diameter. Kermadecia sinuata is found in the central region of New Caledonia mainland and is rare in the north. Kermadecia sinuata grows in dense tropical rainforests above the average elevation of the ground above a somewhat deep sediment substrata. K. sinuata has a pale golden or yellow flower and has a thick and strong inflorescence of 10-45 cm. The tree has stone fruits (40-50 × 25-30 mm) (www.endemia.nc). Asciepias, Milkweed, is an American genus of herbaceous perennial dicotyledonous plants containing more than 140 known species. Asciepias is classified into the subfamily Asclepiadoideae of the Apocynaceae Apocynaceae. Milkweed is named for milk-like sap, which consists of latex containing alkaloid and some other complex compounds including cardenolide. The species of the Asclepias may be found at the following: the species of the Asclepias, the Asclepias, the Asclepias, the Asclipias, the Asclepias, the Ascalulas, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepias, the Asclepis, the Asclepis, the Asclepis, the Asclepis, the Asclepis, the Asclepis, The Eccles. Asclepias linari , Asclepias linearis, Asclepias longifolia, Asclepias meadii, Asclepias nyctaginifolia, Asclepias obovate, Asclepias purpurascens, Asclepias quadrifolia, Asclepias rubra, Asclepias solanoana, Asclepias speciose, Asclepias subulata, Asclepias subverticillata, Asclepias sullivantii, Asclepias syriaca, Asclepias tuberosa, Asclepias uncialis, Asclepias vari Including gate, Asclepias verticillata, Asclepias vestita, Asclepias viridiflora, the Asclepias viridis and Asclepias welshii.

[00144] Cardwellia is a genus of the only described species of large trees, which form part of the bollworm family. The species Cardwellia sublimis (Northern Silky Oak) is endemic to the tropical tropical rainforest of northeastern Queensland, Australia. Other common names include bull oak, golden spanglewood, golden spanglewood, oak and onggaary. This compound has up to 17 leaflets. The compound leaves produce a white inflorescence followed by woody seeds, which are prominently displayed on the outside of the canopy.

[00145] Grevillea is a large genus of trees and shrubs of nearly 340 species of the Phylogenyidae, these species are native to Australia only and few in New Caledonia (Bombarda et al., (2010) J. Am. Oil. Chem. Soc. 87: 981-986; Kato M and Kawakita A. (2004) Am. J. Bot. 91: 1818-1827; Bailey LH and Miller W (1900) Cyclopedia of American Horticulture: Composing Suggestions for Culture of Horticultural Randy Stewart Landscape Design blog-rslandscapedesign.blogspot.com) Plants, Descriptions of the Species of Fruits, Vegetables, Flowers, and Ornamental Plants Sold in the United States and Canada. Most species do not like organic sediments or phosphorus-rich soils. Fertilizers containing phosphorus or potassium can be killed. Pruning after flowering improves vitality. These species are deer resistant, and most species can tolerate severe drought, and many prefer a dry summer. This flower attracts hummingbirds. This species is also used as an edible plant by the larvae of some Lepidoptera species, including dryandra chicks and butterfly (Cyperus sinensis). Phytophora rot is a severe problem for Mediterranean species that overwater during summer. Not only does this species exist in Australia, it has also been found outside of its birth area, such as Italy, where the species is grown.

[00146] Grevillea exul is a small-diameter tree (other sources also call this plant shrub), the largest size to reach is 30 feet, and it is one of the few Grevilleas native to New Caledonia. Grevillea exul is closely related to Grevillea robusta. Its leaves are smooth and not shallow with a maximum length of 9 inches, and the bottom is covered with rusty or silky hair. The white flower blooms in a bunch of toothbrushes long from winter to summer. This plant flourishes on sunny days in well-drained soils of 9-12 cold zones. The variant Grevillea exul var. There is very little information on rubiginosa. G. G. in the presence of Ni salt. exul var. The growth of rubiginosa has been investigated. The seeds are classified as non-dormant (ND) and are fully grown embryos in less than 30 days, water-permeable seed (or fruit) peel. The putative pollinators of this plant are bees or birds. The leaves of subspecies rubiginosa are wider and the bottom is rust red. The flower blooms in a larger bunch.

[00147] G. exul is G. It is close to robusta, and the latter has more information available. Grevillea robusta is a fast-growing, deep-rooted, large, semi-evergreen tree that reaches over 70 feet and is native to southeastern Queensland, Australia. Grevillea robusta is grown as a sunshade in temperate climates worldwide, including Santiago, Chile, California and Florida. Grevillea robusta is highly regarded as a wood plant and is grown commercially in South Africa. That valuable wood is often used to make display cabinets. The wood is not resistant. This species is prosperous in a sunny area of 8b to 12b (CA, TX, LA, FL are considered to be applicable in the United States), prefers a fertile, mainly viscous but well drained soil, and has a mild dryness I need summer. Seedlings are not much colder than mature plants (anpsa.org.au).

[00148] Grevillea decora is a medium upright shrub that is 2 to 5 meters high. The leaves are narrow, pointed or oval and peel grayish green, 7 to 18 cm x 2.5 to 7 cm, with golden red and compact hairy shoots. It is a colorful plant that grows well in arid tropical areas as long as it is well drained soil. This species is not suitable for cold, warm weather. This species breeds best from shredded seeds. Cuttings have been successful but can be difficult to root. This species is G.. It has been successfully grafted on robusta rootstocks and grafted seedlings grow well in relatively cool areas. Some botanists g. It is regarded as a goodii variant.

[00149] Orites is a genus of nine known species of the physalis family. Seven species are from Australia, two from South America, one from Chilean Andes, and one from Bolivia. This species includes Orites acicularis, Orites diversifolius, Orites excellus, Orites fiebrigii, Orites lancifolius, Orites megacarpus, Orites milliganii, Orites myrtoidea and Orites revolutus.

[00150] Orites diversifolius is a common species that extends from the lowland rainforest of western Tasmania to the lower part of the alpine zone (subalpine forest area) (a shrub that grows slowly up to 2 m, resistant to frost and snow). The leaves are quite variable, but usually the width is more than twice the length. Rainforest plants often have leaves approximately 10 cm long, and plants from sub-alpine areas exposed to wind and rain are likely to be about 3-5 cm long. The attractive white flowers of the Ginseng spikelet appear in early summer, but the flowering period may be irregular. Leaves of this type may be full edged, but more often have sharp shallow lobes. The back of the leaf is covered with white powder. This species requires well-composted soil, adequate moisture and a cool place. This species is capable of forming small diameter trees under good conditions. This species grows in partial to full shade (www.utas.edu.au; www.desertnorthwest.com; www.apstas.com; www.australianplants.com).

[00151] Orites revolutus, also known as Narrow-Leaf Orites, is a plant species native to Tasmania. Scottish botanist Robert Brown formally described this species in the London Linne Society newsletter from specimens collected at Lake St Clair in 1810. It is abundant in alpine and subalpine wilderness, and Orites revolutus grows as high bushes or upright woody shrubs, usually 0.5-1.5 m (1 ft 8 in-4 ft 11 in) high. The branches are dense and the leaves alternate with stems. The leaf shape is thin, fairly round at the top, 7-20 mm long, 1-1.5 mm wide, the rim is a tight outer wrap and the back is a hairy surface. Flowering occurs from early summer to midsummer, and sour-smelling flowers rise to twice the length of the leaf in the panicle inflorescence. The color is white, and the flower is 5 mm long, and it has a radial symmetry and is bisexual, with four stamens and upper ovaries. The corolla is valve-like, buds are tubular and split when mature. In fact, it is a fruity bagasse up to 15 mm and contains wing-like seeds. Om. revolutus is likely to provide a substantial food source to nectivorous animal species within the area. The average annual temperature in the area of this plant tends to be around 8 ° C. and rainfall tends to be as low as 1700 or even 2000 mm per year.

[00152] Ziziphus is a genus of the Euphorbiaceae, a genus of about 40 thorny shrubs and small trees of Rhamnaceae, distributed in warm temperate and subtropical regions around the world. Some species are deciduous and others are evergreen. The fruits are edible stone fruits, yellowish brown, red or black spheres or rectangles, 1 to 5 cm (0.39 to 1.97 in) long, often very sweet and sugary Reminiscent of the texture and taste of date palms. The species of the Ziziphus are the following: ziziphus abyssinica, ziziphus angolito, ziziphus apetala, ziziphus attopensis, ziziphus budhensis, ziziphus celata, ziziphus cotinipholia, ziziphus fungihichi ighi ich, html, Ziziphus jujube, Ziziphus laui, Ziziphus lotus, iziphus mairei, Ziziphus mauritiana, Ziziphus melastomoides, Ziziphus Mexicana, Ziziphus mistol, Ziziphus montana, Ziziphus mucronata, Ziziphus nummularia, Ziziphus obtusifolia, Ziziphus oenoplia, Ziziphus oxyphylla, Ziziphus parryi, Ziziphus platyphylla, Ziziphus quadrilocularis, Ziziphus robertsoniana, Ziziphus rugose, Ziziphus saeri , Ziziphus spina-c Including risti, Ziziphus talanai, Ziziphus trinervia, Ziziphus undulata, the Ziziphus xiangchengensis and Ziziphus xylopyrus.

[00153] Ziziphus jujuba is commonly referred to as jujube (sometimes jujuba), red jujube, large jujube, Korean jujube (Korean date), or jujube jujube. Ziziphus jujuba is mainly used as a sunshade and bears fruit. This species is a small deciduous or shrub that reaches 5 to 12 m high and usually has thorny branches. The leaves are shiny green, oval and pointed, 2 to 7 cm wide and 1 to 3 cm wide, with three sharp veins and fine toothed edges at the base. This flower is small, 5 mm wide and has five inconspicuous yellow-green petals. This fruit is an egg-shaped stone fruit that can be eaten, 1.5 to 3 cm deep, smooth green when immature, with an apple-like firmness and taste, brown to purplish black during ripening, and finally Wrinkled, looks like a small date palm. There is one hard core similar to the olive core. Fruit yield is 100-150 g / tree. The planting distance is approximately 10 m. The lipid content in fruits is about 0.06% to 0.10% and in dried fruits is about 1.1%. Jujube fruits have smaller kernels and lower lipid content than jojoba fruits. Thus, in contrast to jojoba, jujubes are not considered as biodiesel feedstocks. Dried fruits and oil extracts are commercially available.

[00154] Hicksbeachia is a genus of trees of the two species of the Phylumidae. These species are native to tropical rain forests north of New South Wales and southeast of Queensland. These seeds are commonly known as red bopple nuts or beef nuts thanks to their bright red colour. The two species are Hicksbeachia pilosa and Hicksbeachia pinnatifolia.

[00155] Hicksbeachia pinnatifolia is a rare species native to New South Wales, Australia, and subtropical rainforests of Queensland. Common names include red bopple nuts, peanuts, red nuts, beef nuts, rose nuts and ivory silky oak. This tree produces thick red fruits between spring and summer. These contain edible seeds. H. Pinnatifolia leaves are bifoliate or winged and 40 to 100 cm long. This plant generally blooms in winter. Red bopple nuts produce thick red fruits in spring and summer. The fruit is eaten, stone-like, egg-shaped, 2 to 4 cm long, non-splitting, thick, red, with flutes on one side, containing large almond sized nuts (nuts). Red bopple nuts have the same taste as macadamia and may be used for similar purposes. This tree carries a relatively large amount of high quality nuts. Although tree propagation depends on seeds, seeds can only survive for a short time. H. Pinnatifolia is difficult to establish because seedlings are prone to various disorders. In reality, poor seedling establishment and poor growth limit the possibilities of this kind (www.fruitandnuttrees.com). This genus is considered similar to but inferior to macadamia.

[00156] Hippophae, Sea buckthorn, is a deciduous shrub of the Gummy family. Hippophae is also called sandthorn, sallowthorn or seaberry. Hippophae rhamnoides, a common sea buckthorn, is by far the most widespread among species of the genus, and its eight subspecies extend from the Atlantic coast of Europe to northwestern Mongolia and northwest China. More than 90%, or about 1,500,000 ha, of the world's natural seabuckthorn habitats are found in China, Mongolia, Russia, Northern Europe and Canada, where the plant blocks soil, water and wildlife protection desertification Used for purpose, as well as for consumer products. Since the species belonging to this genus accumulate lipids in mesocarp, oil can be extracted from the seeds or the flesh of the fruits. The oil content of the seeds of sea buckthorn averages 7 to 11%, and the oil content of pulp is about 1.5 to 3% (per fresh weight). Seed oil is characterized by a high content of polyunsaturated fatty acids, and fruit oil contains monounsaturated fatty acids and carotenoids. Linoleic acid and alpha linolenic acid are the major fatty acids in seed oil, and seabuckthorn flesh oil contains a mixture of palmitoleic acid, which is approximately 65% monounsaturated fatty acid, and palmitic acid, which is a saturated fatty acid. Both oils also contain clogged amounts of tocopherol, tocotrienol and plant sterols. Hippophae species include Hippophae goniocarpa, Hippophae gyantsensis, Hippophae litangensis, Hippophae neurocarpa, Hippophae rhamnoides, Hippophae salicifolia and Hippophae tibetana.

[00157] Ephedra is a genus of gymnosperm shrubs, its family, Maow and the eye, the only genus of Maow. Various species of Maow are spread on many lands and are native to South America South, South Europe, North Africa and South West and Central Asia, North China, and West South America. Common names in English include joint-pine, jointfir, Mormon-tea or Brigham tea. E. Plants of the genus Ephedra, including sinica and others, have traditionally been used by indigenous peoples for various medical purposes, including treatment of asthma, hay fever, and cold. The alkaloid ephedrine and pseudoephedrine are disclosed in E. coli. It is the active ingredient of sinica and other members of this genus. These compounds are sympathostimulatory agents with stimulating and decongestant qualities, and are chemically substituted amphetamines. The species of Maow is Ephedra alata, Ephedra altissima, Ephedra Americana, Ephedra antisyphilitica, Ephedra aphyllica, Ephedra x arenicola, Ephedra aspera, Ephedra aurantiaca, Ephedra boelckei, Ephedra boschantzevii, Ephedra Ephedra coryi, Ephedra cutleri, Ephedra dahurica, Ephedra dawue sis, Ephedra disachya, Ephedra x eleutherolepis, Ephedra equisetina, Ephedra fasciculata, Ephedra fedtschenkoae, Ephedra foemineae, Ephedra foliata, Ephedra fragilis, Ephedra frustilla, Ephedra funereal, Ephedra faces kardangensis, Ephedra khurikensis, Ephedra larista nica, Ephedra likiangensis, Ephedra lomatolepis, Ephedra major, Ephedra milleri, Ephedra minuta, Ephedra monosperma, Ephedra multiflora, Ephedra nevadensis, Ephedra oxyphyata, Ephedra pachyhichidehich, as a portion of a ich Ephedra rhytidosperma, Ephedra rituens s, including Ephedra rupestris, Ephedra sarcocarpa, Ephedra sinica, Ephedra somalensis, Ephedra strobilacea, Ephedra sumlingensis, Ephedra tilhoana, Ephedra torreyana, Ephedra transitoria, Ephedra triandra, Ephedra trifurca, Ephedra tweedieana, the Ephedra viridis and Ephedra vvedenskyi.

[00158] Placospermum is a genus of single species of large trees that form part of the bollworm family. The Placospermum coriaceum species are native to the rainforest of the moist tropical region of northeastern Queensland, Australia. Common names include rose silky oak and plate-seeded oak.

[00159] Xylomelum is a genus of six species of the borage botanical family. Six species are native to Australia and grow in the form of tall shrubs and trees. This genus includes at least two species with the generic name woody pear, Xylomelum pyriforme in the eastern Australian states and Xylomelum occidentales in the western Australian states. The species of Xylomelum includes Xylomelum angustifolium, Xylomelum benthamii, Xylomelum cunninghamianum, Xylomelum occidentale, Xylomelum pyriforme and Xylomelum scottianum.

[00160] Jojoba has the plant name Simmondsia chinensis, but it has goat nuts, goat nuts, deer nuts, pignuts, pig nuts, wild hazels, quinine nuts, coffee tree nuts. Also known as (coffee berry) and gray box bush. Jojoba is native to southwestern North America. Simmondsia chinensis is the only species of the family Simmondsiaceae and is placed on the Caryophyllae eyes. Jojoba is grown commercially to produce jojoba oil, which is a liquid wax ester extracted from its seed. This oil is a very long (C36-C46) linear wax ester and not a triglyceride, and jojoba and its derivatives jojoba ester is rare in that it resembles human sebum and soy sauce more than traditional vegetable oils is there. Jojoba is of interest to the industry as it is odorless and has a viscosity that is temperature independent. Applications vary from engine lubricants to cooking oils. Jojoba wax is predominantly used in medicine, especially in skin products. Its use as a biodiesel fuel is becoming increasingly important. Jojoba oil consists of long linear monoesters of 22 to 44 carbon atoms (as opposed to most vegetable oils consisting of triglycerides), which makes jojoba oil comparable to diesel in terms of energy density.

[00161] Gevuina avellana, also called Hardy macadamia (Hardy Macadamia), chilli nut (Chile Nut), and Spanish in Spanish as Avellano, is native to southern Chile and Argentina, and is rarely cultivated in the northern hemisphere, although it is known None (San B and Yildirim AN (2010) J. Food Compos. Anal. 23: 706-710). Gevuina avellana is a Proteaceae family of evergreen broad-leaved trees, ranging from shrubs to trees 60 feet tall. This plant grows up to approximately 2,300 feet (700 meters) above sea level. The range extends from 35 to 44 degrees south latitude. This plant can be adversely affected by heat and may wither from cold freezing inland North America. This plant can flourish in places such as Ireland, Scotland, relatively warm parts of England, and New Zealand. It is possible to add rich nuts even in independent trees. Nuts can be grown each year. Nuts can be easily peeled off. Analysis shows that the nuts contain about 12.5% protein, about 49.5% oil, and about 24.1% carbohydrate. In Chile, highly mono-saturated oils are also extracted for various uses. The germinated seeds are easy to eat by rodents. The tree is adaptable to highly diverse soil and sun levels. This species does not like phosphorus fertilization (www.arthurleej.com). In some classifications, Gevuina is recognized in three species native to Australia (Gevuina bleasdalei) and New Guinea (Gevuina papuana), respectively, and one species in Chile and Argentina (Gevuina avellana). Other taxonomic reports put Australia and New Guinea species in the genus Bleasdalea or in the genus Trillillia native to Fiji, leaving only Gevuina avellana in Gevuina. The Australian flora keeps these two species at Gevuina, but more recent classifications of Australia and New Guinea species as Bleasdalea bleasdalei and B. edulis. It is regarded as papuana.

[00162] In some embodiments, the plants are Gevuina avellana, Corylus avellana, Kermadecia sinuata, Asclepias syriaca, Cardwellia sublimis, Grevillea exul var. It is selected from the group consisting of rubiginosa, Orites diversifolius, Orites revoluta, Ziziphus jujube, Hicksbeachia pinnatifolia and Grevillea decora.

[00163] In some embodiments, the one or more naturally occurring organisms comprise one or more algae. In various embodiments described herein, one or more algae may be selected from green algae, yellow-green algae and red algae.

[00164] In some embodiments, the one or more algae comprises one or more green algae. In certain embodiments, the one or more green algae comprises a species from the genus Pediastrum. In some embodiments, the one or more green algae comprises Pediastrum simplex. In certain embodiments, the one or more green algae comprises a species from the genus Chlorococcum. In some embodiments, the one or more green algae comprises Chlorococcum isabeliense. In certain embodiments, the one or more green algae comprises a species from the genus Parachlorella. In some embodiments, the one or more green algae comprises Parachlorella kessleri. In certain embodiments, the one or more green algae comprises a species from the genus Oocystis. In some embodiments, the one or more green algae comprises Oocystis heteromucosa. In certain embodiments, the one or more green algae comprises a species from the genus Phacotus. In some embodiments, the one or more green algae comprises Phacotus lenticularis. In certain embodiments, the one or more green algae comprises a species from the genus Dactylococcus. In certain embodiments, the one or more green algae comprises a species from the genus Eremosphaera. In some embodiments, the one or more green algae comprises Eremosphaera viridis. In certain embodiments, the one or more green algae comprises a species from the genus Chlamydomonas. In some embodiments, the one or more green algae comprises Chlamydomonas monticola. In certain embodiments, one or more green algae comprises a species from the genus Dicranochaete. In some embodiments, the one or more green algae comprises Dicranochaete reniformis.

[00165] In some embodiments, the one or more algae comprises one or more yellow-green algae. In certain embodiments, one or more yellow-green algae comprises a species from the genus Heterococcus. In some embodiments, one or more yellow-green algae comprises Heterococcus crassulus. In some embodiments, one or more yellow-green algae comprises Heterococcus pleurococcoides. In certain embodiments, one or more yellow-green algae comprises species from the genus Chlorobotrys. In some embodiments, the one or more yellow-green algae comprises Chlorobotrys regularis.

[00166] In some embodiments, one or more algae comprises one or more red algae. In certain embodiments, the one or more red algae comprises a species from the genus Compsopogon. In some embodiments, the one or more red algae comprises Compsopogon coeruleus.

[00167] In some embodiments, the one or more naturally occurring organisms comprise one or more flagellates. In some embodiments, the one or more flagellates comprise species from the genus Distigma. In some embodiments, the one or more flagellates comprises Distigma curvata. In some embodiments, the one or more flagellates comprise species from the genus Gyropaigne. In some embodiments, the one or more flagellates comprises Gyropaigne lefevrei.

[00168] In some embodiments, one or more naturally occurring organisms comprise one or more diatoms. In some embodiments, one or more diatoms comprise species from the genus Skeletonema. In some embodiments, one or more diatoms include Skeletonema subsalsum.

[00169] In some embodiments, the one or more naturally occurring organisms comprise one or more bacteria. In some embodiments, the bacteria comprises gram negative bacteria. In some embodiments, the gram negative bacteria comprises a species from the genus Myxococcus. In certain embodiments, the gram negative bacteria comprises Myxococcus xanthus.

[00170] In some embodiments, the bacteria comprises cyanobacteria. In some embodiments, the cyanobacteria comprises a species from the genus Synechococcus. In certain embodiments, the cyanobacteria comprises Synechococcus elongatus. In some embodiments, the cyanobacteria comprises a species from the genus Chlorogloeopsis. In certain embodiments, the cyanobacteria comprise Chlorogloeopsis fritschii. In some embodiments, the cyanobacteria comprises a species from the genus Aphanothece. In certain embodiments, the cyanobacteria comprises Aphanothece hegewaldii. In some embodiments, the cyanobacteria comprises a species from the genus Chroococcus. In certain embodiments, the cyanobacteria comprises Aphanothece hegewaldii.

Enzymes and / or proteins introduced into one or more recombinant microorganisms
[00171] The present disclosure is a method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties, comprising one or more unsaturated lipids. The invention relates to methods comprising producing one or more unsaturated lipid moieties in one or more recombinant microorganisms engineered to include a biosynthetic pathway for production of the moieties. In one embodiment, the one or more recombinant microorganisms engineered to include the biosynthetic pathway for the production of one or more unsaturated lipid moieties comprise one or more exogenous biosynthetic enzymes .

Desaturase
[00172] The present disclosure describes enzymes that desaturate fatty acyl substrates to the corresponding unsaturated fatty acyl substrates.

[00173] In some embodiments, desaturases are used to catalyze the conversion of fatty acyl CoA or acyl ACP to the corresponding unsaturated fatty acyl CoA or acyl ACP. Desaturases remove at least two hydrogen atoms to produce the corresponding unsaturated fatty acid / acyl, thereby forming a saturated fatty acid or fatty acid derivative, such as fatty acyl CoA or fatty acyl ACP (herein "aliphatic acyl" The enzyme that catalyzes the formation of a carbon-carbon double bond in Desaturases are classified in terms of the ability of the enzyme to selectively catalyze double bond formation on carbon near the end with respect to the methyl end of fatty acid / acyl or carbon near the end with respect to the carbonyl end of fatty acid / acyl. Omega (ω) desaturases catalyze the formation of a carbon-carbon double bond at a carbon near a certain end to the fatty acid / acyl methyl end. For example, omega ^3-desaturase catalyzes the formation of a double bond between the third and fourth carbon respect methyl end of the fatty acid / acyl. Delta (Δ) desaturases catalyze the formation of carbon-carbon double bonds at specific positions with respect to the carboxyl group of fatty acid or the carbonyl group of fatty acyl CoA. For example, delta ⁹ catalyze the formation of double bonds between C ₉ and C ₁₀ carbon relative to the carboxyl terminus or carbonyl groups of the fatty acyl-CoA fatty acids.

[00174] As used herein, desaturase is described in terms of the position at which it catalyzes the formation of a double bond and the resulting geometric configuration (ie E / Z) of the unsaturated hydrocarbon. It is possible. Thus, as used herein, a Z9 desaturase catalyzes the formation of a double bond between C ₉ and C ₁₀ carbons to the carbonyl end of a fatty acid / acyl, thereby carbon-carbon A Δ desaturase that places two hydrocarbons on opposite sides of a double bond in a cis or Z configuration. Similarly, as used herein, a Z11 desaturase is a Δ desaturase that catalyzes the formation of a double bond between the C ₁₁ and C ₁₂ carbons to the carbonyl end of a fatty acid / acyl. is there.

[00175] Desaturases have a conserved structural motif. This sequence motif of transmembrane desaturases is characterized by [HX3-4HX7-41 (3 non-His) HX2-3 (1 non-His) HHX 61-189 (40 non-His) HX2-3 (1 non-His) HH]. The sequence motif of soluble desaturases is characterized by two occurrences of [D / EEXXH].

[00176] In some embodiments, the desaturase is a fatty acyl CoA desaturase that catalyzes the formation of a double bond with fatty acyl CoA. In some such embodiments, the fatty acyl CoA desaturases described herein comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 The fatty acyl CoA can be utilized as a substrate having a chain length of 20, 21, 22, 23 or 24 carbon atoms. Thus, desaturases used in recombinant microorganisms can be selected based on the length of the substrate.

[00177] In some embodiments, the fatty acyl desaturases described herein can catalyze the formation of a double bond at a desired carbon to terminal CoA on unsaturated fatty acyl CoA . Thus, in some embodiments, a desaturase that catalyzes a double bond insertion at positions 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 with respect to the carbonyl group on fatty acyl CoA Can be selected for use in recombinant microorganisms.

[00178] In some embodiments, the fatty acyl desaturases described herein are such that the resulting unsaturated fatty acyl CoA has a cis or trans (ie, Z or E) geometric configuration. , Can catalyze the formation of double bonds in saturated fatty acyl CoA.

[00179] In some embodiments, the desaturase is a fatty acyl ACP desaturase that catalyzes the formation of a double bond in fatty acyl ACP. In some embodiments, the fatty acyl ACP desaturases described herein are: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, Fatty acyl CoA can be utilized as a substrate having a chain length of 21, 22, 23, or 24 carbon atoms. Thus, desaturases used in recombinant microorganisms can be selected based on the length of the substrate.

[00180] In some embodiments, the fatty acyl ACP desaturases described herein catalyze the formation of a double bond at the desired carbon to the terminal carbonyl on unsaturated fatty acyl ACPs. it can. Thus, in some embodiments, a desaturase that catalyzes a double bond insertion at positions 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 with respect to the carbonyl group on fatty acyl ACP Can be selected for use in recombinant microorganisms.

[00181] In some embodiments, the fatty acyl desaturases described herein are such that the resulting unsaturated fatty acyl ACP has a cis or trans (ie, Z or E) geometric configuration. , Can catalyze the formation of double bonds in saturated fatty acyl CoA.

[00182] In some embodiments, the fatty acyl desaturase is stearyl CoA Δ9 desaturase. In certain embodiments, the stearyl CoA Δ9 desaturase is OLE1. In some embodiments, the stearyl CoA Δ9 desaturase is YGL055W from Saccharomyces cerevisiae. In some embodiments, the stearyl CoA Δ9 desaturase is YALI0C05951 g from Yarrowia lipolytica. In some embodiments, the stearyl CoA Δ9 desaturase is CaO19.12583 from Candida albicans. In some embodiments, the stearyl CoA Δ9 desaturase is CaO 19.5117 from Candida albicans. In some embodiments, the stearyl CoA Δ9 desaturase is CTRG — 05479 from Candida tropicalis.

[00183] In some embodiments, the fatty acyl desaturase is oleoyl CoA Δ12 desaturase. In certain embodiments, the oleoyl CoA Δ12 desaturase is FAD2. In some embodiments, the oleoyl CoA Δ12 desaturase is YALI0B10153g from Yarrowia lipolytica. In some embodiments, the oleoyl CoA Δ12 desaturase is CaO19.118 from Candida albicans. In some embodiments, the oleoyl CoA Δ12 desaturase is CaO 19.7765 from Candida albicans. In some embodiments, the oleoyl CoA Δ12 desaturase is CTRG — 02975 from Candida tropicalis.

[00184] In some embodiments, the fatty acyl desaturase is linoleoyl CoA Δ15 desaturase. In certain embodiments, linoleoyl CoA Δ15 desaturase is FAD3. In some embodiments, the linoleoyl CoA Δ15 desaturase is CaO19.4933 from Candida albicans. In some embodiments, the linoleoyl CoA Δ15 desaturase is CaO19.12399 from Candida albicans. In some embodiments, the stearyl CoA Δ9 desaturase is CTRG — 03583 from Candida tropicalis.

[00185] In one exemplary embodiment, the fatty acyl desaturase is a Z11 desaturase. In various embodiments described herein, the Z11 desaturase, or the nucleic acid sequence encoding it, is of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or It is possible to isolate from organisms of An additional Z11 desaturase, or a nucleic acid sequence encoding it, can be isolated from Bombyx mori, Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphania nitidalis. In an exemplary embodiment, the Z11 desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787, and AY493438. In some embodiments, the nucleic acid sequence encoding the Z11 desaturase from the organism of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized . In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NO: 3, 11, 17 and 19 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 25 from Trichoplusia ni. In another embodiment, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOS: 4 and 9 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 29 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 5 and 16 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 26 and 27 from Thalassiosira pseudonana. In a particular embodiment, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NO: 6, 10 and 23 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 28 from Amyelois transitella. In a further embodiment, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 7, 12, 18, 20 and 24 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 1 from Helicoverpa zea. In some embodiments, the Z11 desaturase is S. coli. It comprises the amino acid sequence set forth in SEQ ID NO: 2 from Inferens. In some embodiments, the Z11 desaturases are GenBank Accession Nos. AF416738, AGH12217.1, AII 21943.1, CAJ43430.2, AF441221, AAF 81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, It includes the amino acid sequences set forth in ABX71630.1 and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, a complete or partial Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of the Z11 desaturase has been replaced with an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 8, 21 and 22. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 36, 37 and 38.

[00186] In another exemplary embodiment, the fatty acyl desaturase is a Z9 desaturase. In various embodiments described herein, the Z9 desaturase, or a nucleic acid sequence encoding it, is isolated from an organism of the species Ostrinia furnacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, or Helicoverpa zea It is possible. In an exemplary embodiment, the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332, AF482906, and AAF81788. In some embodiments, the nucleic acid sequence encoding the Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 13 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 30 from Ostrinia furnacalis. In another embodiment, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 14 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 31 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 15 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 32 from Helicoverpa zea.

Flavoprotein Pyridine Nucleotide Cytochrome Reductase
[00187] The present disclosure describes an enzyme that catalyzes an exchange that reduces the equivalent between a one electron carrier and a two electron carrier nicotinamide dinucleotide. Enzymes: Ferredoxin: NADP + Reductase (FNR), Plant and Fungal NAD (P) H: Nitrate Reductase, NADH: Cytochrome b5 Reductase, NADPH: P450 Reductase, NADPH: Sulfite Reductase, Nitric Oxide Synthase, Phthalate It is possible to include acid dioxygenase reductase, and various other flavoproteins.

[00188] In one embodiment, the flavoprotein pyridine nucleotide cytochrome reductase is a reaction: 2a ferricytochrome b5 + NADH → 2a ferrocytochrome b5 + NAD ++ H + catalyzed NADH: cytochrome B5 reductase.

[00189] NADH: Cytochrome B5 Reductase (CBR) is a flaviprotein oxidoreductase that acts as an electron donor to the ubiquitous electron carrier, cytochrome B5. Cytochrome B5 reductase is involved in a variety of metabolic pathways including, but not limited to, steroid biosynthesis, fatty acid desaturation and elongation, P450 dependent reactions and methemoglobin reduction.

[00190] Cytochrome B5 reductase exists as both a soluble enzyme and a membrane-bound enzyme due to alternative splicing of a single mRNA. The soluble form is mainly present in red blood cells and is involved in the reduction of methemoglobin. The membrane bound form of this enzyme is mainly found in the endoplasmic reticulum and outer mitochondrial membrane, where it is involved in fatty acid desaturation, cholesterol biosynthesis and drug metabolism. In the membrane bound form, the N-terminal residue is myristoylated. Deficiency of this enzyme may result in methemoglobinemia.

[00191] Cytochrome B5 reductase is also known as methemoglobin reductase, NADH cytochrome B5 reductase, NADH methemoglobin reductase, and NADH ferrihemoglobin reductase.

[00192] In one embodiment, the flavoprotein pyridine nucleotide cytochrome reductase is a cytochrome B5 reductase. In some embodiments, the cytochrome B5 reductase is Saccharomyces cerevisiae YIL043C. In another embodiment, the cytochrome B5 reductase is Yarrowia lipolytica YALI0 D04983 g. In some embodiments, the cytochrome B5 reductase is Candida albicans CaO 19.1801. In some embodiments, the cytochrome B5 reductase is Candida albicans CaO19.9367. In some embodiments, the cytochrome B5 reductase is Candida tropicalis CTRG_03865. In some embodiments, the cytochrome B5 reductase is Spodoptera exigua HQ852050. In some embodiments, the cytochrome B5 reductase is Spodoptera exigua JX569756. In some embodiments, the cytochrome B5 reductase is Helicoverpa armigera HQ638220. In some embodiments, the cytochrome B5 reductase is Helicoverpa armigera HQ190046. In another embodiment, the flavoprotein pyridine nucleotide cytochrome reductase is a NADPH-dependent cytochrome P450 reductase (CPR1 or NCP1). In some embodiments, the NADPH-dependent cytochrome P450 reductase is Saccharomyces cerevisiae YHR042W. In another embodiment, the NADPH-dependent cytochrome P450 reductase is Yarrowia lipolytica YALI0D04422 g. In some embodiments, the NADPH dependent cytochrome P450 reductase is Candida albicans CaO 19.10187. In some embodiments, the NADPH-dependent cytochrome P450 reductase is Candida albicans CaO 19.2672. In some embodiments, the NADPH-dependent cytochrome P450 reductase is Candida tropicalis CTRG — 00485. In some embodiments, the NADPH-dependent cytochrome P450 reductase is Spodoptera littoralis JX310073. In some embodiments, the NADPH-dependent cytochrome P450 reductase is Spodoptera exigua HQ 852049. In some embodiments, the NADPH-dependent cytochrome P450 reductase is Helicoverpa armigera HM347785.

[00193] In some embodiments, the cytochrome reductase described herein is used to catalyze the conversion of fatty acyl CoA or acyl ACP to the corresponding unsaturated fatty acyl CoA or acyl ACP.

Elongase
[00194] The present disclosure describes enzymes that extend fatty acid chain length.

[00195] Two different types of fatty acid stretch occur in different organisms. These extension pathways use coenzyme A rather than the fatty acid synthesis system FASI and the acyl carrier protein (ACP) of FASII as an acyl carrier. The first type, mitochondrial fatty acid elongation, is the reversal of fatty acid oxidation. It utilizes acetyl CoA as a substrate and extends the fatty acid chain length on two carbons. This process act primarily short acyl CoA than _{C 16.}

[00196] The second process is the endoplasmic reticulum elongation pathway, which is present in plants, mammals, yeast and other lower eukaryotes. This is a four step reaction, each catalyzed by a separate enzyme. These enzymes are beta-ketoacyl-CoA synthase, beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrogenase and trans-2-enoyl-CoA reductase. This pathway acts mainly using a chain length C ₁₆ or greater acyl CoA than is critical for the generation of very long chain fatty acids. This process utilizes malonyl-CoA rather than acetyl-CoA for chain elongation. The first enzyme that causes condensation of malonyl-CoA and acyl-CoA (beta-ketoacyl-CoA synthase) is also called elongase.

[00197] There are several variants of elongase, depending on the host organism. S. In S. cerevisiae, three different genes encode elongases (ELO1, ELO2 (FEN1) and ELO3 (FEN12)), and these elongases have different substrate specificities. ELO1 prefers relatively short saturated fatty acids (C14-C16), ELO2 prefer longer saturated and monounsaturated fatty acids, ELO3 prefer monounsaturated and polyunsaturated fatty acids. In contrast, Trypanosoma brucei has two elongase genes with relatively short fatty acid specificities (ELO1 has a C4-C10 stretch, ELO2 has a C10-C14 stretch). Orthologues of the three yeast elongase genes are identified in both Plasmodium falciparum and Toxoplasma gondii, and these elongases may have similar substrate specificities to the elongase of yeast.

[00198] In some embodiments, the elongase is It is ELO1 derived from S. cerevisiae (YJL196C). In another embodiment, the elongase is S. It is ELO2 (FEN1) derived from S. cerevisiae (YCR 034 W). In one embodiment, the elongase is CaO19.6343 from Candida albicans. In one embodiment, the elongase is CaO19.13699 from Candida albicans. In one embodiment, the elongase is CTRG 03563 from Candida tropicalis. In another embodiment, the elongase is S. It is ELO3 (FEN12) derived from S. cerevisiae (YLR372W). In one embodiment, the elongase is YALI0B 20196 g from Yarrowia lipolytica. In one embodiment, the elongase is CaO 19.8526 from Candida albicans. In one embodiment, the elongase is CaO 19. 908 from Candida albicans. In one embodiment, the elongase is CTRG — 01179 from Candida tropicalis.

[00199] In addition to fatty acid elongation, fatty acid salvage is also included in this pathway. The fatty acids recovered from the host by the action of acyl CoA binding protein (ACBP) are converted to triacyl glycerides and cholesterol esters by the action of ER localization enzyme diacylglycerol O-acyltransferase (DGAT) and sterol O-acyltransferase, respectively It is possible to do and to store in the fat body. There is strong biochemical evidence to support the acquisition of fatty acids from the host. falciparum and T.I. It exists in gondii.

Thioesterase
[00200] The present disclosure describes enzymes that release free fatty acids from acyl ACP or acyl CoA.

[00201] Acyl ACP thioesterases release free fatty acids from acyl ACPs synthesized from de novo fatty acid biosynthesis. This reaction terminates fatty acid biosynthesis. In plants, fatty acid biosynthesis occurs in plastids and thus requires plastid localized acyl ACP thioesterase. In the vegetative tissues of all plants, the main product of acyl ACP thioesterase is oleic acid (C18: 0), and less, but palmitic acid (C16: 0). The released free fatty acid is reesterified to coenzyme A in the pigment envelope and carried out of the plastid.

[00202] There are two isoforms of acyl ACP thioesterases, FatA and FatB. The substrate specificity of these isoforms determines the chain length and levels of saturated fatty acids in plants. The highest activity of FatA is in the case of C18: 1-ACP. FatA has very low activity against other acyl ACPs as compared to C18: 1-ACP. FatB has the highest activity in the case of C16: 0-ACP. Fat B has significantly higher activity with C18: 1-ACP followed by C18: 0-ACP and C16: 1-ACP. Kinetic studies of FatA and FatB show that its substrate specificity with different acyl ACPs results from Kcat values rather than from Km. The Km values of the two isoforms with different substrates are similar on the micromolar scale. With domain swapping of FatA and FatB, it has been shown that the N-terminus of an isoform determines its substrate specificity. Plants that accumulate mostly medium chain saturated fatty acids in seeds evolved with specialized FatB and / or FatA thioesterases (Voelker T, Kinney AJ (2001) Variations in the biosynthesis of seed-storage lipids Annu Rev Plant Physiol Plant Mol Biol 52: 335-361). For example, lauric acid (12: 0) is the main seed oil in coconut. Correspondingly, medium chain specific acyl ACP thioesterase activity was detected in coconut seeds.

[00203] Fatty acids are often found in cells in the activated form of acyl coA. Acyl-CoA is used in the biosynthesis of many cellular products and components. In plants, acyl CoA is involved in the biosynthesis of membrane lipids, seed storage lipids, waxes, cutins and suberins.

[00204] Acyl-CoA thioesterase hydrolyzes fatty acyl-CoA into free fatty acids. In eukaryotes, this enzyme activity has been detected in the cytosol, ER, mitochondria and peroxisomes. In prokaryotes, this enzyme activity is localized to the periplasm and cytosol. In prokaryotes, acyl CoA thioesterases are involved in catabolism and support growth with fatty acids or conjugated fatty acids as the sole carbon source (Nie L et al., (2008) A novel paradigm of fatty acid beta-oxidation demonstrated by the thioesterase-dependent partial degradation of conjugated linoleic acid that fully supports growth of Escherichia coli. Biochemistry 47 (36): 9618-26). The physiological role of acyl-CoA thioesterases in eukaryotes remains unclear. Studies in animals and yeast provide preliminary clues about the involvement of this enzyme in fatty acid oxidation. ACH2 is the first cloned plant acyl-CoA thioesterase (Tilton GB et al., (2004) Biochemical and molecular characterization of ACH2, an acyl-CoA thioesterase from Arabidopsis thaliana. J Biol Chem 279 (9): 7478- Page 94). This gene is highly expressed in mature tissues rather than in sprouting seedlings where fatty acid beta oxidation mainly occurs. This indicates that the role of ACH2 is not associated with fatty acid oxidation.

[00205] In some embodiments, the acyl ACP thioesterase is FatB from Arabidopsis thaliana (Gene ID AT1G08510). In some embodiments, the acyl ACP thioesterase is FatA from Arabidopsis thaliana (Gene ID AT3G25110).

[00206] In some embodiments, the acyl CoA thioesterase is tesA from Escherichia coli (Gene ID EG 11542). In some embodiments, the acyl CoA thioesterase is ACH2 from Arabidopsis thaliana (Gene ID AT1G01710).

Glycerol-3-phosphate acyltransferase
[00207] The present disclosure provides an acylation reaction at the sn-1 position of glycerol 3-phosphate shown below:
Long-chain acyl CoA + sn-glycerol 3-phosphate → 1-acyl-sn-glycerol 3-phosphate + coenzyme A
Explain the enzyme that catalyzes.

[00208] Glycerol-3-phosphate acyltransferase (GPAT) catalyzes an acylation reaction at the sn-1 position of glycerol 3-phosphate. Plant cells contain three types of GPAT, which are located in chloroplasts, mitochondria and cytoplasm. Enzymes in chlorophyll are soluble, using acyl- (acyl carrier protein) as the acyl donor, enzymes in mitochondria and cytoplasm bound to the membrane and using acyl CoA as the acyl donor (Nishida I et al. , (1993) The gene and the RNA for the precursor to the plastid-located glycerol-3-phosphate acyltransferase of Arabidopsis thaliana. Plant MoI Biol. 21 (2): 267-77; Murata N and Tasaka Y (1997) ) Glycerol-3-phosphate acyltransferase in plants. Biochim Biophys Acta. 1348 (1-2): 10 to 16).

[00209] Eight GPAT genes have been identified in Arabidopsis (Zheng Z et al., (2003) Arabidopsis AtGPAT 1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility Plant Cell 15 (8): 1872-87). GPAT1 was shown to encode a mitochondrial enzyme (Zheng et al., 2003). GPAT4, GPAT5 and GPAT8 have been shown to be essential for cutin biosynthesis (Beisson F et al., (2007) The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis. Plant Cell 19 ( 1): 351-368; Li, Y et al. (2007) Identification of acyltransferase required for cutin biosynthesis and production of cutin with suberin-like monomers. Proc Natl Acad Sci USA 104 (46): 18339-18344). GPAT2, GPAT3, GPAT6 and GPAT7 have not yet been characterized.

[00210] Cytoplasmic GPAT is responsible for the synthesis of triacylglycerol and non-chloroplast membrane phospholipids. Cytoplasmic GPAT is predicted to have substrate selectivity for palmitic acid (C16: 0) and oleic acid (C18: 1). Because these two fatty acids are the most common fatty acids found at the sn-1 position of plant triacylglycerols. Cytoplasmic GPAT was partially purified from avocado (Eccleston VS and Harwood JL (1995) Solubilisation, partial purification and properties of acyl-CoA: glycerol-3-phosphate acyltransferase from avocado (Persea americana) fruit mesocarp. Biochim Biophys Acta 1257 (1): 1 to 10).

[00211] E. membrane-bound glycerol-3-phosphate acyltransferase (PlsB) from E. coli catalyzes the first involved step in phospholipid biosynthesis, followed by the enzyme 1-acylglycerol-3-phosphate O-acyltransferase (PlsC) It is believed that it functions immediately (Kessels JM et al. (1983) Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycophosphate acyltransferase from Escherichia coli. Biochim Biophys Acta 753 (2): 227-235). PlsB is specific for the acylation at position 1 of sn-glycerol 3-phosphate using fatty acyl acyl carrier protein (acyl ACP) or fatty acyl coenzyme A (acyl CoA) thioester as an acyl donor It is possible to form 1-acyl-sn-glycerol 3-phosphate. The endogenously synthesized fatty acids are linked to ACP, and the exogenously added fatty acids are linked to CoA. E. In E. coli phospholipids, the sn-1 position is predominantly occupied by palmitic acid or cis-vaccenate and the sn-2 position is primarily palmitoleic acid or cis succinate. It is believed that this results from the substrate selectivity of the PlsB and PlsC enzymes.

[00212] The plsB gene has been shown to be regulated by stress response regulators such as RNA polymerase, sigma 24 (sigma E) factor and ppGpp (Wahl A et al., (2011) Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol 80 (5): 1260-75 Pls B is part of a protein network for phospholipid synthesis and holo- [acyl carrier protein] (ACP) It interacts with esterase / thioesterase (YbgC) and phosphatidylserine synthase (PssA) to form a complex on the cytoplasmic side of the inner membrane.

[00213] plsB is essential for growth (Baba T et al., (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. MoI Syst Biol. 2: 2006-2008; Yoshimura M. et al. (2007) Involvement of the YneS / YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli. BMC Microbiol 7: 69).

[00214] Site-directed mutagenesis and chemical modification studies have shown catalytically important amino acid residues in PlsB, including invariant histidine residues that are essential for catalysis (Lewin TM et al., (1999) ) Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry 38 (18): 5764-5771. Genetic studies have confirmed that the plsB locus is involved in the formation of multidrug resistant survival cells.

[00215] E. The properties of the E. coli B enzyme were examined in previous studies (Kito M et al., (1972) Inhibition of L-glycerol 3-phosphate acyltransferase from Escherichia coli by cis-9, 10-methylenehexadecanoic acid. J Biochem 71 (1): 99-105; Okuyama H and Wakil SJ (1973) Positional specificities of acyl coenzyme A: glycerol phosphate and acyl coenzyme A: monoacylglycerophosphate acyltransferase in Escherichia coli. J Biol Chem 248 (14): 5197-5205; Kito of phospholipids on the regulatory properties of solubilised and membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichia coli. Biochim Biophys Acta 529 (2): 237-249).

[00216] Glycerol-3-phosphate / dihydroxyacetone phosphate dual substrate specific sn-1 acyltransferase is located in lipid particles and ER, and the step of glycerol-3-phosphate and dihydroxyacetone in lipid biosynthesis Involved in chemical acylation. The most conserved motifs and functionally related residues are directed towards the ER lumen.

[00217] The gene (ACT1) encoding double glycerol-3-phosphate O-acyltransferase (GAT) / dihydroxyacetone phosphate acyltransferase (DHAT) has been identified from Saccharomyces cerevisiae, cloned and biochemically characterized It was attached. In yeast Δgpt1 mutants exhibiting very low GAT / DHAT activity, overexpression of SCT1 through a plasmid vector indicates increased GAT / DHAT activity, and glycerol-3-phosphate O-acyltransferase / acyl dihydroxyacetone phosphate It clearly showed the proposed molecular function as a transferase. GAT / DHAT activity on acyl donors was highest for palmitreoyl-CoA, followed by palmitoyl-CoA, oleoyl-CoA and stearoyl-CoA. SCT1p was localized to the endoplasmic reticulum in the cytosolic membrane, nine minutes. In vivo studies of Δsct1 mutants have actually shown that there is an effect on all four phospholipids, but the observed reduction of 16: 0 fatty acids in the phosphatidyl ethanolamine class is due to other fatty acids, in particular It was offset by an increase of 18: 0 molecular species. Null mutants of SCT1 and GPT2 were synthetically lethal in yeast (Zheng Z and Zou J (2001) The initial step of the glycolipid pathway: identification of glycerol 3-phosphate / dihydroxyacetone phosphate dual substrate acyltransferases in (Saccharomyces cerevisiae. J Biol Chem 276 (45): 41710-41716).

[00218] A gene (GPT2) encoding double glycerol-3-phosphate O-acyltransferase (GAT) / dihydroxyacetone phosphate acyltransferase (DHAT) from Saccharomyces cerevisiae has been identified, cloned and biochemically It was characterized. GPT2 does not contain E. coli in the ΔplsB background lacking GAT / DHAT activity. It was recombinantly expressed in E. coli and showed an increase in GAT activity, but it was not possible to rescue the mutant, presumably due to the imprecise embedding of GPT2 in the membrane. In yeast Δgpt1 mutants exhibiting very low GAT / DHAT activity, overexpression of GPT2 from a plasmid vector indicates increased GAT / DHAT activity, and glycerol-3-phosphate O-acyltransferase / dihydroxyacetone phosphate acyl It clearly showed the proposed molecular function as a transferase. GAT / DHAT activity for acyl donors was highest for oleoyl-CoA, followed by palmitreoyl-CoA, palmitoyl-CoA, and stearoyl-CoA.

[00219] GPT2p was localized to the cytosolic membrane. In vivo studies of Δgpt2 mutants did not reveal any significant effect on the overall fatty acid profile, but a 16: 1 fatty acid reduction in the phosphatidylethanolamine class was observed, which is 16: 0 and 18 : Compensated by the increase of one molecular species. Analysis of the known yeast mutant TTA1 which is deficient in GAT activity showed that the TTA1 GPT2 gene has a missense mutation which is altered by one nucleotide with the conserved motif III representing an acyltransferase. Null mutants of SCT1 and GPT2 were synthetically lethal in yeast (Zheng and Zou 2001).

[00220] In some embodiments, the glycerol-3-phosphate acyltransferase is GPAT from Arabidopsis thaliana (At1g02390). In some embodiments, glycerol-3-phosphate acyltransferase is E. coli. It is PlsB derived from E. coli (Gene ID EG10740). In some embodiments, glycerol-3-phosphate acyltransferase is It is a dual glycerol-3-phosphate O-acyltransferase (GAT) / dihydroxyacetone phosphate acyltransferase (DHAT) SCT1 derived from S. cerevisiae (YBL011 w). In some embodiments, the glycerol-3-phosphate acyltransferase is YALI0C00209 g from Yarrowia lipolytica. In some embodiments, the glycerol-3-phosphate acyltransferase is I503_02577 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CTRG — 02630 from Candida tropicalis. In some embodiments, glycerol-3-phosphate acyltransferase is It is a dual glycerol-3-phosphate O-acyltransferase (GAT) / dihydroxyacetone phosphate acyltransferase (DHAT) GPT2 derived from S. cerevisiae (YKR067w). In some embodiments, the glycerol-3-phosphate acyltransferase is CaO19.5815 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CaO19.13237 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CTRG — 02630 from Candida tropicalis.

Lysophosphatidic acid acyltransferase
[00221] The present disclosure describes enzymes that catalyze the acylation of the sn-2 position of triacylglycerols.

[00222] Membrane-bound 1-acylglycerol-3-phosphate O-acyltransferase encoded by gene plsC catalyzes the second step of phospholipid biosynthesis,
It is believed to function just beside the preceding enzyme glycerol-3-phosphate acyltransferase encoded by the gene plsB (Kessels JM et al., 1983). This enzyme is specific for the acylation of the 1-acyl-sn-glycerol 3-phosphate at the sn-2 position, and an acylacyl carrier protein (acyl ACP) or an acyl coenzyme A (acyl CoA) as a fatty acid donor It is possible to form 1,2-diacyl-sn-glycerol 3-phosphate (phosphatidate, phosphatidic acid). The endogenously synthesized fatty acid is linked to ACP, and the exogenously added fatty acid is linked to CoA (Greenway DL and Silbert DF (1983) Altered acyltransferase activity in Escherichia coli associated with mutations in acyl coenzyme A synthetase. J Biol Chem 258 (21): 13034-13042). E. In E. coli phospholipids, the sn-1 position is mainly occupied by palmitic acid or cis succinate and the sn-2 position is mainly palmitoleic acid or cis succinate. It is believed that this results from the substrate selectivity of PlsB and PlsC enzymes (Rock CO et al., (1981) Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol 3- phosphate. J Biol Chem 256 (2): pages 736-742; Goelz SE and Cronan JE (1980) The positional distribution of fatty acids in Escherichia coli phospholipids is not regulated by sn-glycerol 3-phosphate levels. J Bacteriol 144 ( 1): 462-464).

[00223] Site-directed mutagenesis studies have shown that changing threonine 122 to alanine or leucine alters acyl-CoA substrate specificity (Morand LZ et al., (1998) Alteration of the fatty acid substrate specificity of lysophosphatidate Acylase by site-directed mutagenesis. Biochem Biophys Res Commun 244 (1): 79-84).

[00224] E. In engineered strains of E. coli, overexpression of PlsC and GalU increased the production of glyceroglycolipids (Mora-Buye N et al., (2012) An engineered E. coli strain for the production of glycoglycerolipids. Metab Eng 14 (5): 551-559).

[00225] The plsC gene of Streptococcus pneumoniae is E. coli. It encodes a 1-acylglycerol-3-phosphate acyltransferase homologous to the E. coli enzyme. This gene is cloned and cloned into E. coli. E. coli, and it has been shown that membranes expressing this gene catalyze the expected function (Lu YJ et al., (2006) Acyl-phosphates initiate membranes phospholipid synthesis in Gram-positive pathogens. Mol Cell 23 ( 5): 765-772).

[00226] Plant lysophosphatidic acid acyltransferase (LPAAT) catalyzes the acylation of the sn-2 position of triacylglycerol. The substrate specificity of LPAAT in a given plant species generally determines which fatty acid species are incorporated at the sn-2 position. LPAAT was cloned from corn and meadow foam. Meadowfoam has two LPAAT genes and corn has only one. The enzyme activities of both LAT1 and LAT2 of meadowfoam were confirmed by in vitro assay. Furthermore, LAT1 is E.I. It has been shown that E. coli LPAAT-deficient strains are functionally complemented (Brown AP et al., (2002) Limnanthes douglasii lysophospholipidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli pls C gene. Biochem J 364 (Pt) 3): 795-805).

[00227] LAT1 is a highly selective acyltransferase that uses only 18: 1-CoA as a substrate. LAT2 is not very selective. The highest activity was shown for 22: 1-CoA, followed by 16: 0-CoA and 18: 1-CoA. The substrate specificity of LAT1 and LAT2 is consistent with the proposed role of LAT1 in membrane lipid biosynthesis and LAT2 in storage lipid biosynthesis. Plant cell membranes mainly contain C16 and C18 unsaturated fatty acids, and storage lipids contain a wide range of fatty acids including saturated fatty acids and very long chain unsaturated fatty acids. Protein levels of LAT1 and LAT2 in different plant tissues were detected by the antibody. LAT1 is present in both leaves and developing seeds, LAT2 is detected only in developing seeds. This is again consistent with its proposed role. The role of LAT2 in triacylglycerol biosynthesis was further revealed by transformation of LAT2 in Brassica napus that does not normally contain 22: 1-CoA at the sn-2 position. 22: 1-CoA was inserted at the sn-2 position by transformation of meadowform LAT2 (Lassner MW et al., (1995) Lysophosphophilic acid acyltransferase from meadowfoam mediators insertion of elastic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil. Plant Physiol 109 (4): 1389-1394).

[00228] The viable mutant Saccharomyces cerevisiae strain lacking sphingolipid biosynthesis was utilized to isolate the gene SLC1 and demonstrate that it encodes acyl CoA: lysophosphatidic acid transferase. E. coli classified as 1-acyl-sn-glycerol-3-phosphate acyltransferase Sequence homology with the PLSC protein of E. coli showed similar functions. This putative molecular function of SLC1p is described by E.I. Confirmed by the ability to rescue the ΔplsC mutant of E. coli. A single nucleotide change that changes L glutamine to L-leucine at position 131 transforms substrate selectivity from C16 and C18 fatty acids to C26 fatty acids, which in vivo is a wild-type (SLC1) versus mutant (SLC1-) It could be shown that it is reflected in the corresponding fatty acid composition of 1) (Nagiec MM et al., (1993) A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that has an escherbles an escherichia coli fatty acyltransferase. J Biol Chem 268 (29): 22156-22163).

[00229] E. in vitro assay with recombinantly expressed and purified SLC1p in E. coli revealed substrate selectivity for lysophosphatidic acid and oleoyl CoA, but 1-palmitoyl glycerol 3-phosphate and 1-stearoyl -Sn-glycerol 3-phosphate was also accepted. 2. In vivo of mutants such as Δslc1, Δslc4 (another potential acyl CoA: phosphatidylacyl transferase) and double mutants Δslc1Δslc4 carrying plasmids carrying the SLC1 or SLC4 gene called ΔSLC1 (or 2. ΔSLC4) Studies have shown that SLC1 also promotes the biosynthesis of phosphatidate and phosphatidylinositol and diacylglycerol. It has been suggested that SLC1 is involved in phospholipid remodeling by fatty acid exchange on glycerophospholipids in vivo (Benghezal M et al. (2007) SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1- Acylycerol-3-phosphate O-acyltransferases of budding yeast. J Biol Chem 282 (42): 30845-30855).

[00230] The yeast genome having a candidate open reading frame (ORF) of a known acyltransferase enzyme was screened and the relevant defective strain was tested to identify the gene encoding acyl CoA-dependent lysophospholipid acyltransferase (ALE1) . A dramatic decrease in lysophosphatidylethanolamine acyltransferase (LPEAT) activity was observed in the Δale1 strain, but the major LPAAT in Saccharomyces cerevisiae, ie, when SLC1p is absent or inactive, ALE1p overlaps It could also be demonstrated that it can provide the active lysophosphatidic acid acyltransferase (LPAAT) activity. ALE 1 p preferably binds unsaturated acyl chains of different lengths to the sn-2 position of lysophospholipids. This enzyme has been localized to both microsomal and mitochondrial membranes using high purity cell fractions. It has been proposed that ALE1 could be the primary name LPEAT in the foreign lysolipid metabolism (ELM) pathway in yeast, but ALE1 is also required for the efficient functioning of the endogenous Kennedy pathway (Riekhof WR et al., (2007) Identification and characterization of the major lysophylidyl acetylacylacyltransferase in Saccharomyces cerevisiae. J Biol Chem 282 (39): 28344-28352).

[00231] In a simultaneous study, LPT1 (which is synonymous with ALE1) was identified by applying synthetic gene array analysis and shown to have lysophospholipid acyltransferase activity. In this study, the best substrate for LPT1 (= ALE1) is lysophosphatidylcholine, and thus acts as lysophosphatidylcholine acyltransferase (LPCAT), and the residual activity as previously reported LPAAT is also single Δlpt1 and double Δscl1 Δlpt1 sudden Demonstrated using a mutant, the latter was nonviable. The rate of incorporation of oleic acid into phosphatidylcholine was determined as 70% for de novo synthesis and 30% for remodeling (Jain S et al., (2007) Identification of a novel phosphophosphoridyl acyl transferase in Saccharomyces cerevisiae. J Biol Chem 282 (42): 30562-30569).

[00232] The molecular function of ALE1 (also called LCA1 or SLC4) as lysophosphatidylcholine acyltransferase (LPCAT) is another simultaneous study monitoring the incorporation of radiolabeled lysophosphatidylcholine and / or palmitoyl CoA into phosphatidylcholine (PC) Confirmed by In this study, ALE1p (= LCA1p in this study) accepted various acyl donors, but the highest activity as LPCAT regardless of the acyl chain (16: 0 or 18: 1) of lysophosphatidylcholine species Indicated. Furthermore, concentrations above 0.1 mM were inhibitory and at lower concentrations (10-25 μM) high sensitivity to activating Zn 2+ was observed. The high PC turnover rate measured for ALE1p (= LCA1p) highlighted this enzyme as an important catalyst involved in PC reacylation (Chen Q et al., (2007) The yeast cyclylcerol acyltransferase LCA1 is a key component of Lands cycle for phosphatidylcholine turnover. "FEBS Lett 581 (28): 5511-5516).

[00233] The search for genes causing abnormalities in the formation of lipid droplets (LD) in Saccharomyces cerevisiae has identified the gene LOA1 (formerly VPS 66) encoding the acyl CoA-dependent lysophosphatidic acid acyltransferase. The in vivo molecular function of LOA1p was determined using a comparison of the lipidome of wild type and Δloa yeast strains. Analysis showed that in the LOA1-deficient mutant (Δloa1), the percentage of oleic acid containing phosphatidic acid species was significantly reduced and the content of triacylglycerol (TGA) was reduced by 20% . The protein is E. coli. by recombinant expression in E. coli and obtaining highly concentrated lipid droplet fractions, and partially purified by affinity chromatography with LOA1p which has been permanently attached to the matrix beads. Purified LOA1p was characterized in an in vitro assay, demonstrating that LOA1p is specific for lysophosphatidic acid and oleoyl CoA and thus acts as oleoyl CoA: lysophosphatidic acid acyltransferase in yeast. Based on this result, LOA1p was proposed to be significantly involved in pouring excess oleic acid-containing phosphatidic acid species into TAG biosynthesis and proper growth of lipid droplets (LD). The site of LOA1 could be located in the endoplasmic reticulum (ER) and lipid droplet (LD) using genome tagging constructs, intracellular fractionation, immunohistochemistry and fluorescence microscopy (Ayciriex S et al., (2012) YPR139c / LOA1 encodes a novel lysophosphatidic acid acyl transferase associated with lipid droplets and involved in TAG homeostasis. MoI Biol Cell 23 (2): 233-246).

[00234] In some embodiments, lysophosphatidic acid acyltransferase is E. coli. It is plsC derived from E. coli (MetaCyc accession number ID EG11377). In another embodiment, lysophosphatidic acid acyltransferase is S. It is plsC derived from P. pneumoniae (MetaCyc Accession No. ID G-10763). In some embodiments, the lysophosphatidic acid acyltransferase is LAT1 from Limnanthes douglasii. In some embodiments, the lysophosphatidic acid acyltransferase is LAT2 from Limnanthes douglasii (MetaCyc Accession ID G-9398). In some embodiments, the lysophosphatidic acid acyltransferase is SLC1 from Saccharomyces cerevisiae (YDL052c). In some embodiments, the lysophosphatidic acid acyltransferase is YALI0E18964g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.250 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.7881 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRG — 02437 from Candida tropicalis. In some embodiments, the lysophosphatidic acid acyltransferase is ALE1 from Saccharomyces cerevisiae (YOR175C). In some embodiments, the lysophosphatidic acid acyltransferase is YALI0F19514 g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.1881 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO 19.9437 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRG_01687 from Candida tropicalis. In some embodiments, the lysophosphatidic acid acyltransferase is LOA1 from Saccharomyces cerevisiae (YPR139C). In some embodiments, the lysophosphatidic acid acyltransferase is YALI 0 C 14014 g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.1043 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO 19.8645 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRG — 04750 from Candida tropicalis.

Diacylglycerol acyltransferase
[00235] The present disclosure describes enzymes that add an acyl group to the sn-3 position of diacylglycerol (DAG) to form triacylglycerol (TAG).

[00236] Diacylglycerol acyltransferase (DGAT) catalyzes only one unique reaction in triacylglycerol biosynthesis. This enzyme adds an acyl group at the sn-3 position of diacylglycerol (DAG) to form triacylglycerol (TAG), the reaction shown as follows.

Acyl CoA + 1,2-diacyl-sn-glycerol → triacyl-sn-glycerol + coenzyme A

[00238] As demonstrated for Arabidopsis DGAT, DGAT accepts a broad spectrum of acyl-CoAs, including C18: 1, C18: 2, and C20: 1 acyl-CoAs as acyl donors (Jako C et al., (2001) Seed- Plant Physiol 126 (2): 861-874). Specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhance the seed oil content and seed weight. Expression of DGAT's Arabidopsis cDNA in insect cell cultures and in yeast and overexpression of this cDNA in wild-type Arabidopsis demonstrated DGAT activity in transferring the acyl group to the sn-3 position of DAG (Hobbs DH) (1999) Cloning of a cDNA encoding diacylglyceryl acyltransferase from Arabidopsis thaliana and its functional expression. FEBS Lett 452 (3): 145-149; Zou J et al. (1999) The Arabidopsis thaliana mutants have a mutation Plant J 19 (6): 645-653). Overexpression of Arabidopsis cDNA in wild type Arabidopsis increases oil deposition in seeds, which increase is correlated with increased levels of DGAT mRNA expression. This indicates that DGAT is a regulatory point in the triacylglycerol biosynthetic pathway.

[00239] The gene encoding bifunctional acyl-CoA: acylglycerol acyltransferase (DGAT) was identified as a major contributor to triacylglycerol biosynthesis in Saccharomyces cerevisiae (Sandager L et al., (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277 (8): 6478-6482). This gene (DGA1) belongs to the DGAT2 family, and this member is characterized as an acyl-CoA-dependent acyltransferase (Lardizabal KD et al., (2001) DGAT2 is a new diacylglyceryl acyltransferase gene family: purification, cloning, and expression "J Biol Chem 276 (42): 38862-3869)) DGA1p esterifies diacylglycerol (DAG) into triacylglycerol (TAG) in the yeast genome It has been demonstrated that it is the only acyl CoA-dependent acyltransferase catalyzing the other diacylglycerol acyltransferase important in deletion mutants of DGA1 (Δdga1) and in yeast, ie phospholipid acyl Use donor In combination with the deletion of LRO1 (Δlro1), which esterifies DAG, in the Δdga1 Δlro1 double mutant, almost all of the diacylglycerol acyltransferase is lost and the TAG synthesis is abolished. The carrying plasmid was able to rescue the TAG synthesis defect in the mutant, and in vivo DGA1 was shown to be significantly involved in the TAG biosynthesis route (Sorger D, Daum G (2002) Synthesis) of triacylglycols by the acyl-coenzyme A: diacyl-glyceryl acyltransferase Dgalins in lipid particles of the yeast Saccharomyces cerevisiae J. Bacteriol 184 (2): pp. 519-524; Oelkers P et al. synthetic pathway in yeast. J Biol Chem 277 (11): 8877-8881). A preference for DGA1p over oleoyl CoA and palmitoyl CoA is observed in vitro, and this preference is reversed for the phospholipid-dependent acyltransferase LRO1p (Oelkers et al., 2002).

[00240] Furthermore, the function of DGA1p as an acyl-CoA dependent monoacylglycerol acyltransferase (MGAT) was demonstrated in vivo utilizing a Δdga1 mutant that has lost more than 60% of MGAT activity. The in vitro MGAT activity of DGA1 was demonstrated by oleoyl CoA-dependent esterification of 2-oleoylglycerol to give 1,2-dioleoylglycerol in this process (Heier C et al. (2010) Identification of Yju3p as functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1801 (9): 1063-1071).

[00241] More insights into the functional importance and morphological orientation of sequence motifs in the primary sequence of DGA1p are obtained by in silico analysis, site-directed mutagenesis of signature motifs and deletion mutations at the C and N termini. It was done. In addition to the signature motifs found in other DGAT2 family members, it was possible to demonstrate that Saccharomyces has a unique hydrophilic stretch that has been shown to significantly modulate enzyme activity. Furthermore, the second histidine residue 195 of the four defined transmembrane domains has been shown to be essential for enzyme activity. The topology of DGA1 revealed that both the C and N termini face the cytoplasm and the C termini was found to be more important than the N termini for DGA1 activity (Liu Q et al., (2011 Year) Functional and topological analysis of yeast acyl-CoA: diacylglycerol acyltransferase 2, an endoplasmatic reticulum enzyme essential for triacylglycerol biosynthesis. J Biol Chem 286 (15): 13115 to 13126).

[00242] Using highly purified cell fragments and immunoblots, Sorger et al. (2002) and Liu et al., (2011) identified the position of DGA1 into lipid droplets and microsomal membranes, nine minutes through the endoplasmic reticulum. It proved that it could be traced.

[00243] Acinetobacter sp. ADP1 expresses a bifunctional enzyme that exhibits both wax ester synthase (WS) and acyl coA: diacylglycerol acyltransferase (DGAT) activity (Kalscheuer R and Steinbuchel A (2003) A novel bifunctional wax ester synthase / acyl-CoA: diacylglycerol acyltransferase mediators wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278 (10): 8075-8082). This homodimer catalyzes the final step of TAG and WE biosynthesis (Stoveken T et al., (2005) The wax ester synthase / acyl coenzyme A: diacylglyceryl acyltransferase from Acinetobacter sp. Strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 187 (4): 1369-1376). This homodimer mediates both oxoester and thioester bond formation, has a broad substrate range, and accepts medium chain fatty alcohols and acyl CoA esters as well as monoacylglycerol (MAG) (Uthoff S et al., 2005 ) Thio wax ester biosynthesis utilizing the non-specific bifunctional wax ester synthase / acyl coenzyme A: diacylglyceryl acyltransferase of Acinetobacter sp. Strain ADP1. Appl Environ Microbiol 71 (2): 790-796).

[00244] In some embodiments, the diacylglycerol acyltransferase is TAG1 from Arabidopsis thaliana (Gene ID AT2G19450). In some embodiments, the diacylglycerol acyltransferase is S. It is DGA1 derived from S. cerevisiae (YOR245c). In some embodiments, the diacylglycerol acyltransferase is atfA from Acinetobacter sp. Species ADP1 (MetaCyc Accession No. ID ACIAD 0832). In some embodiments, the diacylglycerol acyltransferase is YALI0E 32769 g from Yarrowia lipolytica. In some embodiments, the diacylglycerol acyltransferase is CaO 19.6941 from Candida albicans. In some embodiments, the diacylglycerol acyltransferase is CaO19.14203 from Candida albicans. In some embodiments, the diacylglycerol acyltransferase is CTRG — 06209 from Candida tropicalis.

[00245] Phospholipid: diacylglycerol acyltransferase (PDAT) catalyzes the following reaction: phosphatidylcholine + 1, 2-diacyl-sn-glycerol → triacyl-sn-glycerol + 1-acyl-sn-glycero-3-phosphocholine.

[00246] Arabidopsis thaliana PDAT can use different phospholipids having an acyl group of 10 to 22 carbon chain length in either sn-position as an acyl donor (Stahl U et al., (2004) Cloning and functional characterization of a phospholipid: diacylglyceryl acyltransferase from Arabidopsis. Plant Physiol 135 (3): 1324 to 1335). However, the acyl group at the sn-2 position of phosphatidylcholine is used three times the sn-1 position. The highest activity is in the case of acyl groups with multiple double bonds, epoxy or hydroxy groups. Among those tested, the enzyme activity was highest for ricinoleoyl. The 18: 0 and 22: 1 acyl groups gave the lowest enzyme activity. Among the different phospholipid species, the higher activity than in the case of phosphatidic acid or phosphatidyl choline is that of phosphatidyl ethanolamine.

[00247] PDAT activity was detected in the seed microsome fraction of castor bean. Radiolabeled ricinoleoyl and vernoloyl groups are effectively transferred from phosphatidylcholine to DAG to form triacylglycerols (Dahlqvist A et al., (2000) Phospholipid: diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent Formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci USA 97 (12): 6487-6492).

[00248] In another embodiment, the diacylglycerol acyltransferase is phospholipid: diacylglycerol acyltransferase (PDAT). In some embodiments, the PDAT is from Arabidopsis thaliana (Gene ID AT5G13640). In some embodiments, the PDAT is from Ricinus communis. In some embodiments, the PDAT is LRO1 from Saccharomyces cerevisiae (YNR008w). In some embodiments, the PDAT is YALI0E16797g from Yarrowia lipolytica. In some embodiments, the PDAT is CaO 19.13439 from Candida albicans. In some embodiments, the PDAT is CTRG_04390 from Candida tropicalis.

[00249] In some embodiments, glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidyl acid acyltransferase (LPAAT), glycerol phospholipid acyltransferase (GPLAT) and / or diacylglycerol acyltransferase (DGAT) One or more genes of the encoding recombinant microorganism are missing or down-regulated and replaced by one or more heterologous GPAT, LPAAT, GPLAT, or DGAT variants. In some embodiments, one or more acyltransferase variants are derived from one or more heterologous acyltransferases selected from Table 3c. In some embodiments, the recombinant microorganism is Y.1. lipolytica YALI0 C00209 g, Y. lipolytica YALI 0 E 18964 g, Y. lipolytica YALI 0 F 19514 g, Y. lipolytica YALI 0 C 14014 g, Y. lipolytica YALI 0 E 16 797 g, Y. lipolytica YALI 0 E 327 69 g, and Y. lipolytica YALI 0 D 0 79 86 g, S. S. cerevisiae YBL011 w, S.I. S. cerevisiae YDL052 c, S.I. S. cerevisiae YOR 175 C, S.I. S. cerevisiae YPR139C, S.I. S. cerevisiae YNR 008 w and S. C.cerevisiae YOR245c and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaR1 9.9437, Candida CaO19. .13439, Candida CTRG_04390, Candida CaO 19.6941, Candida CaO 19.14203 and Candida CTRG_06209; Including deletion, disruption, mutation, and / or reduction of the activity of one or more endogenous acyltransferase enzymes.

[00250]

Lipid binding protein
[00251] The present disclosure describes proteins that are capable of moving steroids, phospholipids and gangliosides between cell membranes.

[00252] Sterol carrier proteins are also known as nonspecific lipid transfer proteins. These proteins are different from plant non-specific lipid transfer proteins, but are structurally similar to small proteins of unknown function derived from Thermus thermophilus. Human sterol carrier protein 2 (SCP2) is a basic protein believed to be involved in the intracellular transport of cholesterol and various other lipids.

[00253] SCP-2 belongs to the SCP-2 gene family including SCP-X, SCP-2, 17β-hydroxysteroid dehydrogenase IV, stomatin, UNC-24, and metallo β-lactamase (vertebrate); It has been identified in many species, including insects, plants, yeast, bacteria and fungi.

[00254] Sterol carrier proteins have been implicated primarily in a wide range of cholesterol / lipid related functions in vertebrates and insects. Recent studies have demonstrated that SCP-2 has cholesterol / lipid binding activity. SCP-2 is capable of binding to cholesterol, palmitic acid, fatty acyl CoA, acidic phospholipids and bile salts. The binding affinity of SCP-2 to cholesterol is most potent among lipids.

[00255] SCP2 is believed to be a soluble sterol carrier because of its ability to bind sterols and its localization to multiple organelles such as peroxisomes, mitochondria, endoplasmic reticulum, and cytosol.

[00256] Multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate in its gene and protein structure. The SCP-x / pro-SCP-2 gene is a fusion gene with separate initiation sites encoding 15 kDa pro-SCP-2 (without enzyme activity) and 58 kDa SCP-x (3-ketoacyl CoA thiolase). Both proteins share the same cDNA and an amino acid sequence of the 13 kDa SCP-2 at its C-terminus. The cell 13 kDa SCP-2 emerges from the complete posttranslational cleavage of 15 kDa pro-SCP-2 and from the partial post translational cleavage of 58 kDa SCP-x. The putative physiological functions of SCP-2 are enhancement of intermembrane lipid transfer (eg cholesterol, phospholipids) and fatty acyl CoA acyl in vitro, in transfected cells and in genetically engineered animals It has been proposed based on the activation of enzymes involved in group transfer (cholesterol ester, phosphatidic acid). At least four important SCP-2 structural domains have been identified and associated with specific functions. First, the 46 kDa N-terminal presequence present at 58 kDa SCP-x is a 3-ketoacyl CoA thiolase specific for branched chain acyl CoA. Second, the N-terminal 20 amino acid presequence of 15 kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 and enhances its intracellular targeting encoded by the C-terminal peroxisomal targeting sequence . Third, the N-terminal 32 amino acids form an amphipathic alpha-helical region, one of which represents the membrane bound domain. The positively charged amino acid residue on one side of the amphiphilic helix allows SCP-2 to bind to the membrane surface containing anionic phospholipids. Fourth, the hydrophobic surface of the N-terminal amphipathic alpha-helix accommodates multiple types of lipids (eg, fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids), along with beta strands 4, 5 and helix D (Stolowich NJ et al. (2002) Sterol carrier protein-2: structure revels function. Cell Mol Life Sci 59 (2): 193-212).

[00257] In one embodiment, the lipid binding protein is a sterol carrier protein. In another embodiment, the sterol carrier protein is Sterol carrier protein-2 (SCP-2) from Homo sapiens (NP_002970). In another embodiment, the sterol carrier protein is sterol carrier protein-2 (SCP-2) from Yarrowia lipolytica (YALI0E01298-CAG78989.1-SCP2). In another embodiment, the sterol carrier protein is a sterol carrier protein from Candida tropicalis (CTRG — 02171-EER 33353.1).

Fatty acid synthase complex
[00258] The present disclosure describes enzymes that catalyze carbon chain elongation in fatty acids.

[00259] In some embodiments, fatty acid synthase complexes are used to catalyze the initiation and elongation of carbon chains in fatty acids. "Fatty acid synthase complex" refers to a group of enzymes that catalyze the initiation and elongation of carbon chains in fatty acids. ACP, along with enzymes in the fatty acid synthase (FAS) pathway, control the length, saturation and branching of the fatty acids produced. The steps in this pathway are catalyzed by fatty acid biosynthesis (fab) and enzymes of the acetyl CoA carboxylase (acc) gene family. Depending on the desired product, one or more of these genes can be attenuated, expressed or overexpressed. In an exemplary embodiment, one or more of these genes are attenuated, deleted, disrupted and / or mutated.

[00260] There are two major classes of fatty acid synthases. The Type I (FASI) system utilizes a single large multifunctional polypeptide and is common to both mammals and fungi (but differs in the structural arrangement of fungal and mammalian synthases). Type I FAS systems are also found in the CMN group of bacteria (Corynebacterium, Mycobacteria, and Nocardia). Type II FAS (FAS II) is characterized by the use of separate monofunctional enzymes for fatty acid synthesis and is found in archaebacteria and bacteria.

[00261] Because the domains of FASI multienzyme polypeptide and FASII enzyme are largely conserved, the mechanisms of FASI and FASII extension and contraction are substantially similar.

[00262] Fatty acids are synthesized by a series of decarboxylative Claisen condensation reactions from acyl-CoA and malonyl-CoA. The steps of this pathway are catalyzed by fatty acid biosynthesis (fab) and enzymes of the acetyl CoA carboxylase (acc) gene family. See, for example, Heath et al., Prog. Lipid Res. 40: 467, 2001, for a description of this pathway. This document is incorporated herein by reference in its entirety. Without being limited by theory, in bacteria acetyl-CoA is carboxylated with acetyl-CoA carboxylase (Acc, a multi-subunit enzyme encoded by four separate genes, accABCD) to form malonyl-CoA. In yeast, acetyl CoA is carboxylated by the yeast equivalent of acetyl CoA carboxylase encoded by ACC1 and ACC2. In bacteria, the malonic acid group is transferred to ACP by malonyl CoA: ACP transacylase (FabD) to form malonyl ACP. In yeast, the malonyl palmityl transferase domain adds malonyl from malonyl CoA to the ACP domain of the FAS complex. A condensation reaction then takes place, and malonyl ACP is mixed with acyl CoA to obtain β-ketoacyl ACP. In this way, the hydrocarbon substrate is stretched by 2 carbons.

[00263] Following elongation, the β-keto group is reduced to a fully saturated carbon chain by the sequential action of ketoreductase (KR), dehydratase (DH), and enol reductase (ER). The extended fatty acid chain is carried between these active sites while remaining covalently linked to the phosphopantetheine conjugate of ACP. First, β-ketoacyl ACPs are reduced by NADPH to form β-hydroxyacyl ACPs. In bacteria, this step is catalyzed by β-ketoacyl ACP reductase (FabG). The equivalent yeast reaction is catalyzed by the ketoreductase (KR) domain of FAS. Next, β-hydroxyacyl ACP is dehydrated to form trans-2-enoyl ACP, which in bacteria is either β-hydroxyacyl ACP dehydratase / isomerase (FabA) or β-hydroxyacyl ACP dehydratase (FabZ) or in yeast It is catalyzed by the dehydratase (DH) domain of FAS. NADPH-dependent trans-2-enoyl ACP reductase I, II or III in bacteria (FabI, FabK and Fab L respectively) and FAS enol reductase (ER) domain in yeast reduce trans-2-enoyl ACP To form an acyl ACP. The following cycle is initiated by condensation of malonyl ACP and acyl ACP by β-ketoacyl ACP synthase I or β-ketoacyl ACP synthase II (FabB and FabF in bacteria, respectively, or beta-ketoacyl synthase (KS) domain in yeast) .

[00264] In some embodiments, fatty acid synthase complexes can be used to catalyze the extension of fatty acyl ACPs into corresponding fatty acyl ACPs that are two carbons stretched relative to the substrate.

[00265] In some embodiments, the acetyl CoA carboxylase is ACC1. In certain embodiments, the acetyl CoA carboxylase is YNR016C from Saccharomyces cerevisiae. In certain embodiments, the acetyl CoA carboxylase is YALI 0 C 11407 g from Yarrowia lipolytica. In certain embodiments, the acetyl CoA carboxylase is CaO 19.7466 from Candida albicans. In certain embodiments, the acetyl CoA carboxylase is CTRG — 01007 from Candida tropicalis. In some embodiments, the fatty acid synthase, beta subunit is FAS1. In a particular embodiment, the fatty acid synthase, β subunit is YKL182W from Saccharomyces cerevisiae. In certain embodiments, the fatty acid synthase, β subunit is YALI 0 B 15059 g from Yarrowia lipolytica. In certain embodiments, the fatty acid synthase, β subunit, is CaO 19.979 from Candida albicans. In certain embodiments, the fatty acid synthase, β subunit, is CaO 19.8594 from Candida albicans. In certain embodiments, the fatty acid synthase, beta subunit, is CTRG — 05241 from Candida tropicalis. In some embodiments, the fatty acid synthase, alpha subunit is FAS2. In certain embodiments, the fatty acid synthase, alpha subunit, is S000006152 from Saccharomyces cerevisiae. In certain embodiments, the fatty acid synthase, alpha subunit is YALI 0 B 19382 g from Yarrowia lipolytica. In certain embodiments, the fatty acid synthase, alpha subunit is CaO19.13370-72 from Candida albicans. In certain embodiments, the fatty acid synthase, alpha subunit is CaO 19.5949-51 from Candida albicans. In certain embodiments, the fatty acid synthase, alpha subunit, is CTRG_02501 from Candida tropicalis.

Pentose phosphate pathway
[00266] The pentose phosphate pathway (PPP) is a central and widely conserved metabolic pathway for carbohydrate metabolism located in the cytoplasm of eukaryotic cells. This pathway serves two major functions: the generation of precursors for the biosynthesis of macromolecules and the generation of reducing equivalents in the form of NADPH. Thus, these two roles are reflected in the two major phases of PPP: in the "oxidation phase", glucose 6-phosphate (G6P), along with the formation of NADPH, 6-phospho D-glucono-1, Glucose-6-phosphate dehydrogenase (ZWF1) which converts to 5-lactone; 6-phosphogluconolactonase (SOL3 which converts 6-phospho D-glucono-1,5-lactone to D-gluconic acid 6-phosphate SOL 40); is converted to ribulose 5-phosphate (Ru5P) through the sequential action of 6-phosphogluconate dehydrogenase (GND1, GND2) which converts D-gluconate 6-phosphate to Ru5P with the formation of NADPH. The “non-oxidation phase” involves the isomerization of Ru5P to ribose 5-phosphate (R5P), the epimerization of Ru5P to xylulose 5-phosphate (X5P), and transketolases (TKL1, TKL2) and transaldolase (transaldolase) Through the action of TAL1, NQM1), pentose phosphate is converted to fructose 6 phosphate (F6P) and glyceraldehyde 3 phosphate (GAP), both of which are glycolytic intermediates, and to erythrose 4-phosphate (E4P) Perform a series of carbon framework transfers that can be interconverted. The net effect of the non-oxidative phase is to easily interconvert the two pools of sugars, creating an equilibrium between the pentoses required for macromolecule biosynthesis and the hexoses required for energy management.

[00267] Oxidative branches are considered to be largely irreversible under normal cellular conditions, and non-oxidative branches are reversible. PPP is not a simple linear path as some carbon atoms are recycled to glycolysis. Furthermore, the enzyme transketolase catalyzes two different reactions in this pathway, and the substrates of these reactions become competitive inhibitors of one another. PPP is three major products: reducing equivalents in the form of the NADPH, which are produced in the oxidation phase and are required in the biosynthetic pathway and for the maintenance of cellular oxidation levels; R5P for the biosynthesis of all nucleic acids; E4P for biosynthesis of aromatic amino acids. Different physiological states require the action of this biochemical network in different ways: in actively growing cells, such as during culture growth in a reactor, this pathway is sufficient for three products You have to generate everything. Because everything is needed to construct new cells. Under stress conditions, growth is slowed and the only product with considerable demand is NADPH.

An enzyme that is expressed to increase the level of one or more coenzymes
[00268] Nicotinamide adenine dinucleotide (including NAD, NAD + and NADH) and nicotinamide adenine dinucleotide phosphate (including NADP, NADP + and NADPH) have energy metabolism, mitochondrial function, calcium homeostasis, antioxidant property / It belongs to the basic common mediators of various biological processes, including the development of oxidative stress, gene expression, immune function, senescence, and cell death: NAD mediates energy metabolism and mitochondrial function; NADH-dependent reactive oxygen species (ROS) generation from mitochondria and NADPH oxidase-dependent ROS generation are two crucial mechanisms of ROS generation; generated from NAD and NADP Cyclic ADP-ribose and some other molecules NAD and NADP regulate multiple key factors in cell death such as mitochondrial permeability transition, energy status, poly (ADP-ribose) polymerase-1, and apoptosis inducer; and NAD and NADP Significantly affect factors affecting aging such as oxidative stress and mitochondrial activity, and NAD-dependent sirtuins also mediate the aging process. Furthermore, in situ regeneration of reduced nicotinamide cofactor (NAD (P) H) is necessary for the practical synthesis of many important chemicals in recombinant microorganisms.

[00269] The present disclosure describes enzymes that catalyze the simultaneous formation of oxidized and reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate (NAD (P) H) for a given substrate. Several enzyme components are involved in the maintenance of the NADP and NADPH pools. NAD kinase (NADK) catalyzes the direct phosphorylation of NAD to NADP and thus contributes to the generation of cellular NADP pool (Pollak N et al. (2007) The power to reduce: pyridine nucleotides-small molecules with Biochem. J. 402: pp. 205-218; Anglel L et al. (2010) The phosphate makes a difference: cellular functions of NADP. Redox Rep. 15: 2-10). On the other hand, ferredoxin NADP reductase (FNR) in photosynthetic cells in bright period is recognized as a major source of NADPH. However, in non-photosynthetic cells during the dark period of photosynthesis, the main enzymes capable of producing a power reduction in the form of NADPH are: glucose-6-phosphate dehydrogenase (G6 PDH, EC 1.1. 1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) (both belong to the pentose phosphate pathway), NADP isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42) And NAD- or NADP malate enzyme (NADP-ME, EC 1.1.1.40), also known as NAD- or NADP malate dehydrogenase.

[00270] In Saccharomyces cerevisiae, the interconversion of various NAD / NADP species is paramount to maintain cellular redox balance, which in turn determines growth efficiency and metabolite output ( Marres CA et al., (1991) Isolation and inactivation of the nuclear gene encoding the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae. Eur J Biochem 195 (3): 857-62). Various pyridine nucleotides, in particular NADH, can not cross the inner membrane of mitochondria. For that reason, direct cross exchange of cytosol to mitochondria and vice versa of NAD + and reduced and / or phosphorylated derivatives is not possible. In addition, yeast does not have transhydrogenase. Thus, yeast can not convert NAD + and NADPH directly to NADP + and NADH (Bruinenberg PM (1986) The NADP (H) redox ox in couple metabolism. Antonie Van Leeuwenhoek 52 (5): 411-29). As a result, in order to ensure the availability of NAD + metabolites required by each organelle, yeast has developed an independent set of reciprocal exchange reactions that provide all the NAD + types through various enzymatic reactions.

[00271] In the cytoplasm, two NAD kinases that phosphorylate NAD + have been characterized. The major cytosolic NAD kinase is encoded by UTR1 (Kawai S et al., (2001) Molecular cloning and identification of UTR1 of a yeast Saccharomyces cerevisiae as a gene encoding an NAD kinase. F EMS Microbiol Lett 200 (2): 181- 184; Shi F et al. (2005) Identification of ATP-NADH kinase isozymes and their contribution to supply of NADP (H) in Saccharomyces cerevisiae. FEBS J 272 (13): 3337-3349). A second less important NAD kinase for phosphorylation of NAD +, YEF1, has also been described and shown to compensate for the loss of UTR1 (Shi et al., 2005). Deletion of both mitochondrial POS5 and cytosolic UTR1 is lethal in yeast and YEF1 can compensate only when POS5 is still functional. These findings support the notion that yeast's major NADH kinases, namely POS5 and UTR1, are able to partially compensate for each other's loss of activity (Bieganowski P et al., 2006 And J) Biol Chem 281 (32): 22439 to 22445) Synthetic lethal and biochemical analytes of NAD and NADH kinases in Saccharomyces cerevisiae establishment separation of cellular functions. It has been shown that both UTR1 and YEF1 exhibit NADH kinase activity and provide a cytosolic set of enzymes with catalytic ability to phosphorylate NADH.

[00272] Various enzymatic reactions have been shown to explain the provision of NADPH to the cytosol required for biosynthetic pathways and antioxidant function. ALD6 encoded cytosolic acetaldehyde dehydrogenase (Meaden PG et al., (1997) The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic, Mg (2 +)-activated acetaldehyde dehydrogenase. Yeast 13 (14): 1319 to 1327; Wang X, et al. (1998) Molecular cloning, characterization, and potential roles of cytosolic and mitochondrial aldehydes in ethanol metabolism in Saccharomyces cerevisiae J. Bacteriol 180 (4): 822-830) and β-D-glucose 6 phosphate Glucose-6-phosphate dehydrogenase (ZWF1) (Nogae I and Johnston M (1990) Isolation and characterization of the catalysis catalyzes the first step of the pentose phosphate pathway converting to 6-phospho D-glucono-1,5-lactone ZWF1 gene of Saccharomyces cerevisiae, encoding glucose-6-phosphate dehydrogenase. Gene 96 (2): 161 to 169; Grabowska D and Chelstowska A (2003) The ALD6 gene product is in J. Biol Chem 278 (16): 13984-13988; Minard KI and McAlister-Henn L (2001) Antioxidant function of cytosolic sources of NADPH in yeast. Free Radic Biol Med 31 (6): 832-843) is considered as a major supplier of NADPH. The third enzyme that produces NADPH in the cytoplasm is the cytosolic NADP-dependent isocitrate dehydrogenase (IDP2) (Loftus TM et al., (1994) Isolation, characterization, and disruption of the yeast gene encoding cytosolic NADP-specific isocyanate dehydrogenase Biochemistry 33 (32): 966- 9667; Contreras-Shannon V et al. (2005) Kinetic properties and metabolic contributions of yeast mitochondrial and cytosolic NADP +-specific isolate dehydrogenases. J Biol Chem 280 (6): 4469-4 ). ZWF1 has been demonstrated to have overlapping functions with isocitrate dehydrogenase (IDP2) and acetaldehyde oxidoreductase (ALD6), and has been shown to compensate for NADPH deficiency if one of the reactions involved can not operate (Minard KI and McAlister-Henn L (2005) Sources of NADPH in yeast with carbon source. J Biol Chem 280 (48): 39890-39896).

[00273] Enzyme systems currently available for NAD (P) H regeneration are formate / formate dehydrogenase for NADH, isopropanol / alcohol dehydrogenase (Pseudomonas sp.) For NADH, isopropanol / alcohol dehydrogenase for NADPH (T. brockii), H2 / hydrogenase (M. thermoautotrophicum) for both NADH and NADPH, glucose-6-phosphate / glucose-6-phosphate dehydrogenase (L. mesenteroides) for NADPH and NADH and NADPH (Hummel W (1999) Large-scale applications of NAD (P) -dependent oxidoreductases: Recent developments. Trends Biotechnol. 17: 487-492; Wichmann R and Vasic-Racki). D (2005) Cofactor regeneration at the lab scale. Adv Biochem Eng Biote chnol 92: 225-260). Engineering of a highly stable and active mutant phosphate dehydrogenase from Pseudomonas stutzeri and evaluation of its potential as an effective NADPH regenerating system has also been described (Johannes TW et al., (2007) Efficient regeneration of NADPH using an engineered phosphatase dehydrogenase. Biotechnology and Bioengineering 96 (1): 18 to 26).

[00274] In one embodiment, one or more recombinant microorganisms are engineered to increase intracellular levels of coenzyme. In another embodiment, the coenzyme is NADH and / or NADPH. In some embodiments, one or more recombinant microorganisms express a dehydrogenase.

[00275] In certain embodiments, one or more recombinant microorganisms express glucose-6-phosphate dehydrogenase. In some embodiments, glucose-6-phosphate dehydrogenase is ZWF1 from yeast. In another embodiment, the glucose-6-phosphate dehydrogenase is ZWF1 (YNL241C) from Saccharomyces cerevisiae. In another embodiment, glucose-6-phosphate-1-dehydrogenase is zwf from bacteria. In certain embodiments, glucose-6-phosphate-1-dehydrogenase is purified from E. coli. It is zwf (NP_416366) derived from E. coli.

[00276] In certain embodiments, one or more recombinant microorganisms express 6-phosphogluconate dehydrogenase. In some embodiments, the 6-phosphogluconate dehydrogenase is GND1 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND1 (YHR183W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is GND2 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND2 (YGR256W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is gnd derived from bacteria. In certain embodiments, the 6-phosphogluconate dehydrogenase is It is gnd (NP_416533) derived from E. coli.

[00277] In certain embodiments, one or more recombinant microorganisms express NADP + -dependent isocitrate dehydrogenase. In some embodiments, the NADP + dependent isocitrate dehydrogenase is IDP2 from yeast. In some embodiments, the NADP + dependent isocitrate dehydrogenase is YALI0_F04095 g from Yarrowia lipolytica. In another embodiment, the NADP + dependent isocitrate dehydrogenase is CaO 19.3733 from Candida albicans. In certain embodiments, the NADP + dependent isocitrate dehydrogenase is CTRG — 00909 from Candida tropicalis. In some embodiments, the NADP + dependent isocitrate dehydrogenase is YLR174W from Saccharomyces cerevisiae.

[00278] In certain embodiments, one or more recombinant microorganisms express NAD + or NADP + dependent malate dehydrogenase (malate enzyme). In one embodiment, malate dehydrogenase is NAD + dependent. In another embodiment, malate dehydrogenase is NADP + dependent. In one embodiment, the malate enzyme is a bacterial NAD + -dependent malate dehydrogenase. In some embodiments, the NAD + -dependent malate dehydrogenase is It is maeA (NP — 415996) derived from E. coli. In some embodiments, the NAD + -dependent malate dehydrogenase is maeE from Lactobacillus casei (CAQ 68119). In another embodiment, the malate dehydrogenase is a mitochondrial NAD + -dependent malate dehydrogenase from yeast. In some embodiments, the NAD + -dependent malate dehydrogenase is It is MAE1 (YKL029C) derived from S. cerevisiae. In another embodiment, the malate dehydrogenase is a mitochondrial NAD + -dependent malate dehydrogenase from a parasitic nematode. In some embodiments, the NAD + -dependent malate dehydrogenase is M81055 from Ascaris suum. In one embodiment, the malate dehydrogenase is a bacterially derived NADP + dependent malate dehydrogenase. In some embodiments, the NADP + -dependent malate dehydrogenase is It is maeB (NP_416958) derived from E. coli. In one embodiment, the malate dehydrogenase is a NADP + dependent malate dehydrogenase from maize. In some embodiments, the NADP + dependent malate dehydrogenase is me1 from Zea mays.

[00279] In certain embodiments, one or more recombinant microorganisms express aldehyde dehydrokinase. In one embodiment, the aldehyde dehydrokinase is NAD + dependent. In another embodiment, the aldehyde dehydrokinase is NADP + dependent. In one embodiment, the aldehyde dehydrokinase is a bacterial NAD + -dependent aldehyde dehydrokinase. In some embodiments, the NAD + -dependent aldehyde dehydrokinase is E. coli. It is aldA (NP_415933) derived from E. coli. In another embodiment, the aldehyde dehydrokinase is a cytosolic NADP + dependent aldehyde dehydrokinase from yeast. In some embodiments, the NADP + dependent aldehyde dehydrokinase is S. It is ALD6 (YPL061W) derived from S. cerevisiae. In another embodiment, the aldehyde dehydrokinase is cytosolic NADP + dependent aldehyde dehydrokinase from bacteria. In some embodiments, the NADP + dependent aldehyde dehydrokinase is E. coli. It is aldB (NP_418045) derived from E. coli.

[00280] In certain embodiments, one or more recombinant microorganisms express transhydrogenase. In one embodiment, the expression of transhydrogenase is increased to interconvert NADH and NADPH. In some embodiments, the transhydrogenase is a pyridine nucleotide transhydrogenase. In some embodiments, the pyridine nucleotide transhydrogenase is from bacteria. In certain embodiments, the pyridine nucleotide transhydrogenase is E. coli. pntAB derived from E. coli (beta subunit: NP_416119; alpha subunit: NP_416120). In some embodiments, the pyridine nucleotide transhydrogenase is from human. In certain embodiments, the pyridine nucleotide transhydrogenase is NNT from Homo sapiens (NP_036475). In certain embodiments, the pyridine nucleotide transhydrogenase is from Solanum tuberosum. In certain embodiments, the pyridine nucleotide transhydrogenase is from Spinacea oleracea.

[00281] In some embodiments, one or more recombinant microorganisms express glucose 1-dehydrogenase. In some embodiments, the glucose-1-dehydrogenase is from bacteria. In certain embodiments, the glucose-1-dehydrogenase is GOX2015 from Gluconobacter oxydans (Gluconobacter suboxydans) (Q5FPE5). In certain embodiments, the glucose-1-dehydrogenase is gdh from Sulfolobus solfataricus (O93715). In a particular embodiment, the glucose-1-dehydrogenase is gdh from Bacillus subtilis (P12310).

[00282] In some embodiments, one or more recombinant microorganisms express glycerol dehydrogenase. In a particular embodiment, the glycerol dehydrogenase is NADP + dependent glycerol 2-dehydrokinase gld2 from Hypocrea jecorina (Trichoderma reesei) (Q0GYU4). In certain embodiments, the glycerol dehydrogenase is NADP + dependent glycerol dehydrokinase gldB from Emericella nidulans (Aspergillus nidulans) (Q7Z8L1).

[00283] In some embodiments, one or more recombinant microorganisms express alcohol dehydrogenase. In one particular embodiment, the alcohol dehydrogenase is a NADPH dependent alcohol dehydrogenase YMR318C from Saccharomyces cerevisiae. In certain embodiments, the alcohol dehydrogenase is alcohol dehydrogenase CAETHG_0553 from Clostridium autoethanogenum.

[00284] In some embodiments, the alcohol dehydrogenase from Table 3 is used for regeneration of NAD (P) H.

[00285] In some embodiments, one or more recombinant microorganisms express a combination of the above enzymes to increase intracellular levels of coenzyme. In one embodiment, overexpression of the enzyme is linked to complementation of the enzyme's co-substrate to increase intracellular levels of coenzyme.

[00286]

Acyl-CoA oxidase
[00287] Acyl-CoA oxidase (ACO) acts on CoA derivatives of fatty acids having a chain length of 8-18. Acyl-CoA oxidase is a flavin enzyme that contains one non-covalently associated FAD per subunit and belongs to the same superfamily as mitochondrial acyl-CoA dehydrogenase. Like mitochondrial fatty acyl-CoA dehydrogenase, peroxisomal acyl-CoA oxidase catalyzes the initiation and rate-limiting steps of the peroxisomal fatty acid β-oxidation pathway, ie, α, β dehydrogenation of acyl-CoA, and trans-2-enoyl CoA in the reduction half reaction It occurs. In the oxidative half reaction of peroxisomal acyl CoA oxidase, the reduced FAD is reoxidized by molecular oxygen to produce hydrogen peroxide.

[00288] Acyl-CoA oxidase is a homodimer, and the polypeptide chain of subunits is folded into an N-terminal alpha domain, a beta domain, and a C-terminal alpha domain. The functional differences between peroxisomal acyl CoA oxidase and mitochondrial acyl CoA dehydrogenase are attributed to structural differences in the FAD environment.

[00289] In some embodiments, recombinant microorganisms and methods are provided for the production of one or more unsaturated lipid moieties. In certain embodiments, one or more unsaturated lipid moieties have a carbon chain length of C18 or less. In some embodiments, short chain unsaturated lipid moieties are produced from long chain unsaturated lipid moieties. Examples of suitable chain shortening enzymes include FAD-dependent acyl CoA oxidase. For fatty acid molecules with an even number of carbons, chain shortening enzymes produce one molecule of acetyl-CoA and fatty acyl-CoA truncated by two carbons. Fatty acid molecules having an odd number of carbons are oxidized in a similar manner, producing acetyl CoA molecules for each round of oxidation until the carbon chain length is reduced to 5 carbons. In the final cycle of oxidation, this 5-carbon acyl CoA is oxidized to form acetyl-CoA and propionyl-CoA.

[00290] It is known that acyl CoA oxidases show varying specificity for substrates of different chain lengths (Figure 32). Thus, controlling the degree of fatty acyl-CoA cleavage depends on engineering or selecting the appropriate enzyme variant. Examples of acyl CoA oxidases suitable for this purpose are listed in Table 3a.

[00291] In some preferred embodiments of recombinant microorganisms and methods for the production of one or more unsaturated lipid moieties, removal or reduction of cleavage of the desired fatty acyl CoA beyond the desired chain length As such, one or more genes of the microbial host encoding the acyl CoA oxidase are deleted or downregulated. Such deletion or downregulation targets are Y. lipolytica POX1 (YALI0E32835 g), Y.1. lipolytica POX2 (YALI0F10857g), Y.1. lipolytica POX3 (YALI0D24750 g), Y.1. lipolytica POX 4 (YALI0E27654 g), Y.1. lipolytica POX5 (YALI0 C23859 g), Y.1. lipolytica POX 6 (YALI0E06567 g); S. cerevisiae POX1 (YGL205W); Candida POX2 (CaO 19.1655, CaO 19.9224, CTRG_02374, M18259), Candida POX4 (CaO 19.1652, CaO 19.9221, CTRG_02377, M12160) and Candida POX5 (CaO 19.5723, CaO19. , M12161), but is not limited thereto. In other embodiments, one or more endogenous acyl-CoA oxidase enzymes that are deleted, destroyed, mutated, or downregulated control the chain length of one or more unsaturated lipid moieties Do.

[00292]

Acyl glycerol lipase and sterol esterase
[00293] In some embodiments, recombinant microorganisms and methods are provided for the production of one or more short chain unsaturated lipid moieties. In certain embodiments, one or more short chain unsaturated lipid moieties have a carbon chain length of C18 or less. In some preferred embodiments of the method of producing short chain pheromone, selected enzymes that prefer to hydrolyze ester bonds of long chain acylglycerols are coexpressed with one or more fatty acyl desaturases . A good example of such a suitable enzyme is a heterologous or engineered acylglycerol lipase. Examples of acylglycerol lipases suitable for this purpose are listed in Table 3b.

[00294] Carboxyester hydrolases (EC 3.1.1.-) are a large class of enzymes that catalyze the hydrolysis or synthesis of ester bonds. Carboxyester hydrolases have been described in all biological areas of prokaryotes and eukaryotes. Most of the carboxy ester hydrolases belong to the α / β hydrolase superfamily, are formed by His, acidic amino acids and Ser residues and have a conserved “catalytic triad residue” located in the highly conserved GXSXG sequence. During hydrolysis, the catalytic Ser initiates the nucleophilic attack of the substrate with the help of two other residues from the three residues, which are in close spatial proximity. These residues are postulated to facilitate the hydrolysis of the ester by a mechanism similar to that of chymotrypsin-like serine proteases. Another characteristic feature is the presence of an oxyanion hole, an amino acid region whose sequence is not as conserved as that of the catalytic triad, which serves to stabilize the transition states generated in the catalyst. Furthermore, these enzymes generally do not require cofactors. Acyl glycerol lipases and sterol esterases belong to the carboxyester hydrolase family.

[00295] The acylglycerol lipase enzyme catalyzes a chemical reaction that uses water molecules to break down glycerol monoesters of long chain fatty acids. The phylogenetic name of this enzyme class is glycerol ester acyl hydrolase. Common other names include monoacylglycerol lipase, monoacylglycerolipase, monoglyceride lipase, monoglyceride hydrolase, fatty acyl monoester lipase, monoacylglycerol hydrolase, monoglyceryl lipase, and monoglycerylidase. This enzyme is involved in glycerolipid metabolism.

[00296] Sterol esterase enzymes catalyze chemical reactions.

[00297] Steryl ester + H2O <=> sterol + fatty acid

[00298] Thus, two substrates of this enzyme are steryl ester and H2O, and the two products are sterols and fatty acids.

[00299] The phylogenetic name of this enzyme class is steryl ester acyl hydrolase. Common other names include cholesterol esterase, cholesteryl ester synthase, triterpenol esterase, cholesteryl esterase, cholesteryl ester hydrolase, sterol ester hydrolase, cholesterol ester hydrolase, cholesterol, and acyl cholesterol lipase. This enzyme is involved in bile acid biosynthesis. Sterol esterases are widespread in nature and have been identified from mammalian tissues such as pancreas, intestinal mucosa, liver, placenta, aorta, and brain to filamentous fungi, yeasts and bacteria.

[00300] In terms of substrate specificity, many sterol esterases contain ester linkages such as acylglycerols, allyl esters and, in some cases, alcohol esters, cinnamyl esters, xanthophyll esters, or synthetic polymers It can catalyze the hydrolysis or synthesis of a fairly wide range of other substrates.

[00301]

[00302] In some embodiments, one or more acyl glycerol lipase or sterol ester esterase is absent, disrupted, mutated and / or reduced in activity in a recombinant microorganism. In some embodiments, the triacylglycerol lipase that is deleted, destroyed, mutated and / or reduced in activity is YALI 0D 17534 g (TGL 3).

Recombinant microorganism
[00303] The present application is one or more unsaturated lipid moieties, which are converted by non-biological means into one or more fatty alcohols and / or one or more fatty aldehydes Or a recombinant microorganism having a biosynthetic pathway for the production of multiple unsaturated lipid moieties.

[00304] In one embodiment, one or more recombinant microorganisms use one or more carbon sources for the production of one or more unsaturated lipid moieties. In another embodiment, the one or more carbon sources include sugars, glycerol, ethanol, organic acids, alkanes, and / or fatty acids. In certain embodiments, one or more carbon sources are selected from the group consisting of alkanes and fatty acids. In some embodiments, one or more recombinant microorganisms are highly resistant to alkanes and / or fatty acids. In some embodiments, one or more recombinant microorganisms accumulate lipids.

[00305] The present disclosure provides microorganisms that can be engineered to express various foreign enzymes. In one embodiment, one or more recombinant microorganisms are engineered to include a biosynthetic pathway for the production of one or more unsaturated lipid moieties.

[00306] In some embodiments, the recombinant microorganism is one or more desaturases, one or more acyl CoA oxidases, one or more acyl glycerol lipases, one or more flavoprotein pyridine nucleotide cytochromes It comprises one or more exogenous biosynthetic enzymes selected from a reducing enzyme, one or more elongases, one or more thioesterases, and / or one or more lipid binding proteins. In another embodiment, the one or more desaturases are selected from the group consisting of fatty acyl CoA desaturases and fatty acyl ACP desaturases. In some embodiments, one or more flavoprotein pyridine nucleotide cytochrome reductase is selected from the group consisting of cytochrome b5 reductase and NADPH-dependent cytochrome P450 reductase. In some embodiments, the one or more elongases are selected from the group consisting of ELO1, ELO2, ELO3, or any combination thereof. In some embodiments, one or more thioesterases are selected from the group consisting of acyl ACP thioesterases and acyl CoA thioesterases. In some embodiments, one or more lipid binding proteins are sterol carrier protein 2 (SCP2). In some embodiments, one or more of the exogenous biosynthetic enzymes are Saccharomyces cerevisiae, Yarrowia, Candida, Helicoverpa, Agrotis, Trichoplusia, Spodoptera, Ostrinia, Amyelois, Lobesia, Cydia, Grapholita, Lampronia, Sesamia, Plodia, Bombyx , Phoenix, Rattus, Arabidopsis, Plutella and Bicyclus are from a genus selected from the group consisting of In some embodiments, the one or more exogenous biosynthetic enzymes are Saccharomyces cerevisiae, Yarrowia lipolytica, Candida albicans, Candida tropicalis, Candida viswanathii, Helicoverpa armigera, Helicoverpa zea, Agrotis segetum, Trichopos exigua, Amyelois transitella, Lobesia botrana, Cydia pomonella, Lampronia capitella, Grapholita molesta, Ostrinia scapulalis, Ostrinia furnacalis, Ostrinia nubilalis, Ostrinia latipennis, Ostrinia ovalipennis, Plodia interpunctella, Sesamia inferens, Bombyx mori, Phoenix dactylifera, Rattus norvegicus thallium thallium

[00307] In some embodiments, the fatty acyl desaturase comprises fatty acyl CoA 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 Desaturase, which can be used as a substrate having a chain length of 22, 23, or 24 carbon atoms.

[00308] In some embodiments, the fatty acyl desaturase is doubled at the C5, C6, C7, C8, C9, C9, C10, C11, C12, or C13 position of a fatty acid or a derivative thereof, such as, for example, fatty acid CoA ester. It can create bonds.

[00309] In an exemplary embodiment, the fatty acyl desaturase is a Z11 desaturase. In various embodiments described herein, the Z11 desaturase, or the nucleic acid sequence encoding it, is of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or It is possible to isolate from organisms of Additional Z11 desaturases, or nucleic acid sequences encoding them, can be isolated from Bombyx mori, Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphania nitidalis. In an exemplary embodiment, the Z11 desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787 and AY493438. In some embodiments, the nucleic acid sequence encoding the Z11 desaturase from the organism of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized . In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NO: 3, 11, 17 and 19 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 25 from Trichoplusia ni. In another embodiment, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOS: 4 and 9 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 29 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 5 and 16 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 26 and 27 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NO: 6, 10 and 23 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 28 from Amyelois transitella. In a further embodiment, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 7, 12, 18, 20 and 24 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 1 from Helicoverpa zea. In some embodiments, the Z11 desaturase is It comprises the amino acid sequence set forth in SEQ ID NO: 2 from Inferens. In some embodiments, the Z11 desaturases are GenBank Accession Nos. AF416738, AGH12217.1, AII 21943.1, CAJ43430.2, AF441221, AAF 81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, It contains the amino acid sequences described in ABX71630.1 and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, a complete or partial Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of the Z11 desaturase has been replaced with an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 8, 21 and 22. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 36, 37 and 38.

[00310] In a specific embodiment, the Z11 desaturase is Z11-13: acyl CoA, E11-13: acyl CoA, (Z, Z) -7, 11-13: acyl CoA, Z11-14: acyl CoA, E11-14: Acyl-CoA, (E, E) -9, 11-14: Acyl-CoA, (E, Z) -9, 11-14: Acyl-CoA, (Z, E) -9, 11-14: Acyl CoA, (Z, Z) -9, 11-14: Acyl CoA, (E, Z) -9, 11-15: Acyl CoA, (Z, Z) -9, 11-15: Acyl CoA, Z11-16 : Acyl CoA, E11-16: Acyl CoA, (E, Z) -6, 11-16: Acyl CoA, (E, Z) -7, 11-16: Acyl CoA, (E, Z) -8, 11 -16: acyl CoA, (E, E) -9, 11-1 : Acyl CoA, (E, Z) -9, 11-16: Acyl CoA, (Z, E) -9, 11-16: Acyl CoA, (Z, Z) -9, 11-16: Acyl CoA, ((E) E, E) -11, 13-16: acyl CoA, (E, Z) -11, 13-16: acyl CoA, (Z, E) -11, 13-16: acyl CoA, (Z, Z)- 11, 13-16: Acyl-CoA, (Z, E) -11, 14-16: Acyl-CoA, (E, E, Z) -4, 6, 11-16: Acyl-CoA, (Z, Z, E) -7, 11, 13-16: Acyl CoA, (E, E, Z, Z) -4, 6, 11, 13-16: Acyl CoA, Z11-17: Acyl CoA, (Z, Z) -8, 11-17: Acyl CoA, Z11-18: Acyl CoA, E11-18: Acyl CoA, (Z, Z) -1 , 13-18: acyl CoA, (E, E) -11,14-18: it catalyzes the conversion of fatty acyl CoA to Acyl-CoA or single or polyunsaturated product combinations thereof.

[00311] In another exemplary embodiment, the fatty acyl desaturase is a Z9 desaturase. In various embodiments described herein, the Z9 desaturase, or nucleic acid sequence encoding the same, is isolated from an organism of the species Ostrinia furnacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, or Helicoverpa zea It is possible. In an exemplary embodiment, the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332, AF482906, and AAF81788. In some embodiments, the nucleic acid sequence encoding the Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 13 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 30 from Ostrinia furnacalis. In another embodiment, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 14 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 31 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises the nucleotide sequence set forth in SEQ ID NO: 15 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises the amino acid sequence set forth in SEQ ID NO: 32 from Helicoverpa zea.

[00312] In certain embodiments, the Z9 desaturase is Z9-11: Acyl CoA, Z9-12: Acyl CoA, E9-12: Acyl CoA, (E, E) -7, 9-12: Acyl CoA, (E, Z) -7, 9-12: Acyl CoA, (Z, E) -7, 9-12: Acyl CoA, (Z, Z) -7, 9-12: Acyl CoA, Z9-13: Acyl CoA, E9-13: Acyl CoA, (E, Z) -5, 9-13: Acyl CoA, (Z, E) -5, 9-13: Acyl CoA, (Z, Z) -5, 9-13 : Acyl-CoA, Z9-14: Acyl-CoA, E9-14: Acyl-CoA, (E, Z) -4, 9-14: Acyl-CoA, (E, E) -9, 11-14: Acyl-CoA, (E , Z) -9, 11-14: acyl CoA, (Z, E) -9, 11-14: Syl-CoA, (Z, Z) -9, 11-14: Acyl-CoA, (E, E) -9, 12-14: Acyl-CoA, (Z, E) -9, 12-14: Acyl-CoA, (Z , Z) -9, 12-14: Acyl-CoA, Z9-15: Acyl-CoA, E9-15: Acyl-CoA, (Z, Z) -6, 9-15: Acyl-CoA, Z9-16: Acyl-CoA, E9 -16: Acyl-CoA, (E, E) -9, 11-16: Acyl-CoA, (E, Z) -9, 11-16: Acyl-CoA, (Z, E) -9, 11-16: Acyl-CoA , (Z, Z) -9, 11-16: Acyl-CoA, Z9-17: Acyl-CoA, E9-18: Acyl-CoA, Z9-18: Acyl-CoA, (E, E) -5, 9-18: Acyl CoA, (E, E) -9, 12-18: Acyl CoA, (Z, Z)- , 12-18: Acyl-CoA, (Z, Z, Z) -3, 6, 9-18: Acyl-CoA, (E, E, E) -9, 12, 15-18: Acyl-CoA, (Z, Z) , Z) -9, 12, 15-18: Catalyzing the conversion of fatty acyl-CoA into mono- or polyunsaturated products selected from Acyl-CoA, or combinations thereof.

[00313] In some embodiments, the fatty acyl desaturase is E5-10: Acyl CoA, E7-12: Acyl CoA, E9-14: Acyl CoA, E11-16: Acyl CoA, E13-18: Acyl-CoA, Z7-12: Acyl CoA, Z9-14: Acyl CoA, Z11-16: Acyl CoA, Z13-18: Acyl CoA, Z8-12: Acyl CoA, Z10-14: Acyl CoA, Z12-16: Acyl CoA, Z14 -18: Acyl CoA, Z7-10: Acyl CoA, Z9-12: Acyl CoA, Z11-14: Acyl CoA, Z13-16: Acyl CoA, Z15-18: Acyl CoA, E7-10: Acyl CoA, E9- 12: Acyl CoA, E11-14: Acyl CoA, E13-16: Acyl CoA, E15-18: Syl-CoA, E5Z7-12: Acyl-CoA, E7Z9-12: Acyl-CoA, E9Z11-14: Acyl-CoA, E11Z13-16: Acyl-CoA, E13Z15-18: Acyl-CoA, E6E8-10: Acyl-CoA, E8E10-12: Acyl CoA, E10 E 12-14: Acyl CoA, E 12 E 14-16: Acyl-CoA, Z 5 E 8-10: Acyl CoA, Z 7 E 10-12: Acyl CoA, Z 9 E 12-14: Acyl CoA, Z 11 E 14-16: Acyl CoA, Z 13 E 16-18: Acyl CoA, Z3-10: Acyl CoA, Z5-12: Acyl CoA, Z7-14: Acyl CoA, Z9-16: Acyl CoA, Z11-18: Acyl-CoA, Z3 Z5-10: Acyl CoA, Z5 Z7-12: Acyl CoA, 7Z9-14: Acyl-CoA, Z9Z11-16: Acyl-CoA, Z11Z13-16: Acyl-CoA and Z13Z15-18: Catalyzing the conversion of fatty acyl-CoA to mono- or polyunsaturated intermediates selected from Acyl-CoA .

[00314] In some embodiments, the recombinant microorganism can express a bifunctional desaturase that can catalyze the subsequent desaturation of the two double bonds.

[00315] In some embodiments, the recombinant microorganism is a saturated _C 6 _{-C 24} corresponding mono- or polyunsaturated _C 6 _{-C 24} fatty acyl desaturase that catalyzes the conversion of fatty acyl CoA fatty acyl CoA And more than one exogenous nucleic acid molecule encoding. For example, the recombinant microorganism can express an exogenous nucleic acid molecule encoding a Z11 desaturase and another exogenous nucleic acid molecule encoding a Z9 desaturase.

[00316] In some embodiments, one or more recombinant microorganisms engineered to include a biosynthetic pathway for the production of one or more unsaturated lipid moieties have a given cycle producing one or polyunsaturated _C 6 _{-C 24} fatty acyl-CoA substrate as compared to a cut of 2 carbons mono- or polyunsaturated _C 4 _{-C 22} fatty acyl-CoA intermediates start of a cycle, after one or more successive cycles of the acyl-CoA oxidase activity, catalyzes the conversion of one or polyunsaturated fatty acyl CoA single or multivalent truncated forms of unsaturated C ₆ -C ₂₄ fatty acyl CoA And at least one exogenous nucleic acid molecule encoding an acyl CoA oxidase. In some embodiments, the acyl CoA oxidase is selected from Table 3a.

[00317] In some embodiments, the recombinant microorganism is preferably an acylglycerol lipase and / or sterol ester esterase enzyme that hydrolyzes an ester bond of an acylglycerol substrate of> C16,> C14,> C12 or> C10. Further comprising at least one endogenous or exogenous nucleic acid molecule encoding In some embodiments, the expressed acylglycerol lipase and / or sterol ester esterase enzyme is selected from Table 3b.

[00318] In one embodiment, one or more recombinant microorganisms engineered to include a biosynthetic pathway for the production of one or more unsaturated lipid moieties comprises one or more unsaturated lipids. It is further engineered to lack, destroy, mutate, and / or reduce its activity in one or more endogenous proteins in a pathway that competes with the biosynthetic pathway for production of the moiety.

[00319] In some embodiments, the recombinant microorganism lacks, destroys, mutates, and / or one or more endogenous proteins involved in one or more undesired fatty acid elongation pathways. And / or be manipulated to reduce its activity. In certain embodiments, one or more endogenous proteins involved in one or more undesired fatty acid elongation pathways are beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydratase, enoyl-CoA reductase, or It is selected from the group consisting of arbitrary combinations thereof.

[00320] In another embodiment, the recombinant microorganism is a mutation that lacks, destroys, one or more endogenous proteins involved in one or more fat body storage and / or recycling pathways. And / or manipulated to reduce its activity. In certain embodiments, one or more endogenous proteins involved in one or more fat body storage and / or recycling pathways are triacylglycerol lipase, lysophosphatidic acid acyltransferase, steryl ester hydrolase , Acyl CoA synthetase, or any combination thereof. In some embodiments, the triacylglycerol lipase that is missing, destroyed, mutated and / or whose activity is reduced is YALI 0D 17534 g (TGL 3). In some embodiments, the fatty acyl CoA synthetase (fatty acid transporter) that is missing, destroyed, mutated and / or whose activity is reduced is YALI0E16016 g (FAT1).

[00321] In some embodiments, the recombinant microorganism lacks, destroys, mutates, and / or / or one or more endogenous proteins involved in one or more fatty acid degradation pathways. It is manipulated to reduce its activity. In certain embodiments, the fatty acid degradation pathway is a beta oxidation pathway. In certain embodiments, the one or more endogenous proteins are selected from the group consisting of acyl CoA oxidase, multifunctional enoyl CoA hydratase-β-hydroxyacyl-CoA dehydrogenase, β ketoacyl CoA thiolase, or any combination thereof Be done. In some preferred embodiments, one or more genes of the microbial host encoding the acyl CoA oxidase are absent or eliminated to eliminate or reduce cleavage of the desired fatty acyl CoA beyond the desired chain length Down-regulated. In some embodiments, the recombinant microorganism is Y.1. lipolytica POX1 (YALI0E32835 g), Y.1. lipolytica POX2 (YALI0F10857g), Y.1. lipolytica POX3 (YALI0D24750 g), Y.1. lipolytica POX 4 (YALI0E27654 g), Y.1. lipolytica POX5 (YALI0 C23859 g), Y.1. lipolytica POX 6 (YALI0E06567 g); S. cerevisiae POX1 (YGL205W); Candida POX2 (CaO 19.1655, CaO 19.9224, CTRG_02374, M18259), Candida POX4 (CaO 19.1652, CaO 19.9221, CTRG_02377, M12160) and Candida POX5 (CaO 19.5723, CaO19. M12161), deletion, disruption, mutation, and / or reduction of its activity of one or more endogenous acyl CoA oxidase enzymes selected from the group consisting of

[00322] In some embodiments, glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerol phospholipid acyltransferase (GPLAT) and / or diacylglycerol acyltransferase (DGAT) One or more genes of the encoding recombinant microorganism are missing or down-regulated and replaced with one or more heterologous GPAT, LPAAT, GPLAT, or DGAT variants. In some embodiments, one or more acyltransferase variants are derived from one or more heterologous acyltransferases selected from Table 3c. In some embodiments, the recombinant microorganism is Y.1. lipolytica YALI0 C00209 g, Y. lipolytica YALI 0 E 18964 g, Y. lipolytica YALI 0 F 19514 g, Y. lipolytica YALI 0 C 14014 g, Y. lipolytica YALI 0 E 16 797 g, Y. lipolytica YALI 0 E 327 69 g and Y. lipolytica YALI 0 D 0 79 86 g, S. S. cerevisiae YBL011 w, S.I. S. cerevisiae YDL052 c, S.I. S. cerevisiae YOR 175 C, S.I. S. cerevisiae YPR139C, S.I. S. cerevisiae YNR 008 w and S. C.cerevisiae YOR245c and Candida I503_02577, Candida CTRG_02630, Candida CaO 19.250, Candida CaO 19.7.881, Candida CTRG_02437, Candida CaO 19.1881, Candida CaR 19 018, Candida CaO 19 ci. .13439, Candida CTRG_04390, Candida CaO 19.6941, Candida CaO 19.14203 and Candida CTRG_06209 selected from the group consisting of It includes the loss, destruction, mutation and / or reduction of the activity of one or more endogenous acyltransferase enzymes.

[00323] In certain embodiments, the fatty acid degradation pathway is an omega oxidation pathway. In certain embodiments, one or more endogenous proteins are selected from the group consisting of monooxygenase (CYP 52), fatty alcohol oxidase, fatty alcohol dehydrogenase, alcohol dehydrogenase, fatty aldehyde dehydrogenase, or any combination thereof . In some embodiments, the recombinant microorganism is Ylipolytica YALI0E25982 g (ALK1), Y. lipolytica YALI0 F01320 g (ALK2), Y.1. lipolytica YALI0 E23474 g (ALK3), Y.1. lipolytica YALI0 B13816 g (ALK4), Y.1. lipolytica YALI0B 13838 g (ALK5), Y.1. lipolytica YALI 0 B 01 848 g (ALK 6), Y. lipolytica YALI0A 15488 g (ALK7), Y.1. lipolytica YALI 0 C 12122 g (ALK 8), Y. lipolytica YALI0B06248 g (ALK9), Y.1. lipolytica YALI 0 B 20 702 g (ALK 10), Y. lipolytica YALI 0 C 10054 g (ALK 11) and Y. and L. loss, destruction, mutation, and / or reduction of the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI 0 A 20130 g (ALK 12).

[00324] In other embodiments, the recombinant microorganism lacks, destroys, mutates, and / or reduces the activity of one or more endogenous proteins involved in peroxisome biogenesis Operated by In certain embodiments, one or more endogenous proteins involved in peroxisomal biogenesis are PEX1, PEX2, PEX3, PEX4, PEX5, PEX7, PEX7, PEX8, PEX10, PEX11, PEX12, PEX13, PEX14. , PEX 15, PEX 17, PEX 18, PEX 21, PEX 22, PEX 25, PEX 27, PEX 28, PEX 29, PEX 30, PEX 31, PEX 32, PEX 32, or any combination thereof.

[00325] In another embodiment, one or more recombinant microorganisms are further engineered to increase intracellular levels of coenzyme. In certain embodiments, the coenzyme is NADH and / or NADPH. In some embodiments, one or more recombinant microorganisms are glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase, transketolase, ribose-5 -Consists of ketol isomerase, D-ribulose-5-phosphate 3-epimerase, NADP + dependent isocitrate dehydrogenase, NAD + or NADP + dependent malate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, transhydrogenase or any combination thereof It is further engineered to express one or more endogenous or exogenous proteins selected from the group. In another embodiment, expression of one or more endogenous or exogenous proteins is linked to complementation of a co-substrate. In some embodiments, one or more recombinant microorganisms are glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase, transketolase, ribose-5 -Lacking, destroying, mutating, and / or one or more endogenous proteins selected from the group consisting of ketol isomerase, phosphate, D-ribulose-5-phosphate 3-epimerase, or any combination thereof And / or be further manipulated to reduce its activity.

[00326] In some embodiments, the recombinant microorganism expresses one or more acyl CoA oxidase enzymes and lacks, destroys, mutates one or more endogenous acyl CoA oxidase enzymes. And / or are engineered to reduce its activity. In some embodiments, the one or more acyl CoA oxidase enzymes being expressed are different from the one or more endogenous acyl CoA oxidase enzymes that are missing or downregulated. In other embodiments, the expressed one or more acyl CoA oxidase enzymes modulate the chain length of one or more unsaturated lipid moieties. In another embodiment, the one or more acyl CoA oxidase enzymes being expressed are selected from Table 3a.

[00327] In various embodiments described herein, the recombinant microorganism is a eukaryotic microorganism. In some embodiments, the eukaryotic microbe is a yeast. In an exemplary embodiment, the yeast is a member of the group consisting of: .

[00328] In some embodiments, one or more recombinant microorganisms accumulate lipids. In an exemplary embodiment, the recombinant microorganism of the invention is an oleaginous yeast. In a further embodiment, the oleaginous yeast is a member of the group selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. In a further embodiment, the oleaginous yeast is Yarrowia lipolytica, Candida viswanathii, Rhodosporidium toruloides, Lipomyces starkey, L. et al. Lipoferus, C.I. revkaufi, C.I. pulcherrima, C.I. utilis, C.I. T. tropicalis, Rhodotorula minuta, Trichosporon pullans, T. et al. cutaneum, Cryptococcus curvatus, R. et al. glutinis and R.A. It is a member of the species selected from graminis.

[00329] In some embodiments, the recombinant microorganism is a prokaryotic microorganism. In an exemplary embodiment, the prokaryotic microorganism is a member selected from the group consisting of Escherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium and Brevibacterium is there.

[00330] In some embodiments, recombinant microorganisms are used to produce one or more unsaturated lipid moieties.

[00331] Thus, in another aspect, the present invention provides methods of producing one or more unsaturated lipid moieties using recombinant microorganisms as described herein. In one embodiment, the method comprises cultivating the recombinant microorganism in a culture medium containing a feedstock that provides a carbon source until one or more unsaturated lipid moieties are produced. In further embodiments, one or more unsaturated lipid moieties are recovered. Recovery can be by methods known in the art such as distillation, membrane based separation gas stripping, solvent extraction, and expanded bed adsorption.

[00332] In some embodiments, the feedstock comprises a carbon source. In various embodiments described herein, the carbon source may be selected from sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocelluloses, proteins, carbon dioxide, and carbon monoxide. In a further embodiment, the sugar is selected from the group consisting of glucose, fructose and sucrose.

[00333] In some embodiments, one or more recombinant microorganisms are highly resistant to alkanes and / or fatty acids.

[00334] In another aspect, a recombinant microorganism for use in any of the methods of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is provided. Disclosed herein.

Method of producing fatty alcohol and / or aldehyde
[00335] As noted above, in one aspect, a method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is one or more natural methods. Obtaining one or more unsaturated lipid moieties from the organisms present in the as well as directly reducing the one or more unsaturated lipid moieties and reducing to one or more fatty alcohols and / or one or more And a step of producing fatty aldehydes of

[00336] In another aspect, the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is one or more naturally occurring Obtaining one or more unsaturated lipid moieties from the organism; chemically converting one or more unsaturated lipid moieties to one or more free fatty acids (FFAs); one or more FFAs Esterifying one or more fatty acid alkyl esters (FAAE); and reducing one or more FFAs and / or one or more FAAEs, and reduction to one or more fatty alcohols and / or Including the step of producing one or more fatty aldehydes. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[00337] In another aspect, the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties is one or more naturally occurring Obtaining one or more unsaturated lipid moieties from the organism; chemically converting one or more unsaturated lipid moieties to one or more FAAEs; and reducing one or more FAAEs , The reduction of which produces one or more fatty alcohols and / or one or more fatty aldehydes. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[00338] In one embodiment, the one or more naturally occurring organisms are insects. In another embodiment, the one or more naturally occurring organisms are not insects. In some embodiments, one or more naturally occurring organisms are selected from the group consisting of plants, algae and bacteria. In some embodiments, the plant is from a family selected from the group consisting of pokeweed family and buckthorn family. In a particular embodiment, the genus Gevuina, Calendula, Limnanthes, Lunaria, Carum, Daucus, Coriandrum, Macadamia, Myristica, Licania, Aleurites, Ricinus, Corylus, Kermadecia, Asclepias, Grevillea, Orites, Ziziphus, Hicks beach Including species from Ephedra, Plasospermum, Xylomelum and / or Simmondsia. In some embodiments, the plants are Gevuina avellana, Corylus avellana, Kermadecia sinuata, Asclepias syriaca, Cardwellia sublimis, Grevillea exul var. It is selected from the group consisting of rubiginosa, Orites diversifolius, Orites revoluta, Ziziphus jujube, Hicksbeachia pinnatifolia and Grevillea decora. In some embodiments, the algae comprises green algae. In certain embodiments, the green algae comprises a species from the genus Pediastrum. In some embodiments, the green algae comprises Pediastrum simplex. In some embodiments, the bacteria comprises gram negative bacteria. In some embodiments, the gram negative bacteria comprises a species from the genus Myxococcus. In certain embodiments, the gram negative bacteria comprises Myxococcus xanthus. In some embodiments, the bacteria comprises cyanobacteria. In some embodiments, the cyanobacteria comprises a species from the genus Synechococcus. In certain embodiments, the cyanobacteria comprises Synechococcus elongatus.

[00339] In another aspect, a method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties comprises one or more unsaturated lipid moieties Producing one or more unsaturated lipid moieties in one or more recombinant microorganisms engineered to include a biosynthetic pathway for the production of a; one or more unsaturated lipid moieties; Chemically converting to or more free fatty acids (FFA) and / or one or more fatty acid methyl esters (FAAE); and reducing one or more FFAs and / or one or more FAAE Including the step of reducing to produce one or more fatty alcohols and / or one or more fatty aldehydes. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[00340] Any of the methods of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein, comprising a chemical conversion step In one embodiment, chemically converting the one or more unsaturated lipid moieties to one or more FFAs and / or one or more FAAEs comprises one or more unsaturated lipid moieties. Isolating; optionally concentrating one or more unsaturated lipid moieties having a specific chain length; and treating one or more unsaturated lipid moieties to produce a fatty acid derivative Further comprising the step of In some embodiments, the step of treating one or more unsaturated lipid moieties comprises saponification of one or more unsaturated lipid moieties to one or more FFAs. In some embodiments, treating the one or more unsaturated lipid moieties comprises esterification of the one or more unsaturated lipid moieties to one or more FAAEs. In one embodiment, the optional step of concentrating one or more unsaturated lipid moieties having a specific chain length comprises a separation method. In another embodiment, the separation method comprises distillation and / or chromatography. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[00341] In any one embodiment of the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein, The reduction of one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs to one or more fatty alcohols and / or one or more fatty aldehydes Contacting one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs with one or more stoichiometric reducing agents. In some embodiments, the one or more stoichiometric reducing agents are sodium bis (2-methoxyethoxy) aluminum hydride, Vitride (Red-Al, Vitride, SMEAH) and / or hydrogenated diisobutylaluminum (DIBAL) )including. In certain embodiments, the step of reducing one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs comprises bulky cyclic nitrogen Lewis to modify the reducing agent. Contains a base. In one embodiment, the bulky cyclic nitrogen Lewis base provides the selectivity of the reduction step for the production of one or more fatty aldehydes. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[00342] In any one embodiment of the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein, The step of reducing one or more unsaturated lipid moieties, one or more FFAs and / or one or more FAAEs to one or more fatty alcohols comprises one or more unsaturated lipid moieties, one Contacting one or more FFAs and / or one or more FAAEs with one or more transition metal catalysts. In certain embodiments, the one or more transition metal catalysts comprise one or more Group VIII catalysts. In one embodiment, FAAE comprises fatty acid methyl ester (FAME).

[00343] In any one embodiment of the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein, The produced fatty alcohol is further oxidized to fatty aldehyde by the oxidation process. In some embodiments, the oxidation step comprises one or more partial oxidation methods. In certain embodiments, one or more partial oxidation methods include NaOCI / TEMPO. In another embodiment, the oxidation step comprises copper catalyzed aerobic oxidation.

[00344] In any one embodiment of the method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein, The produced one or more fatty alcohols are further chemically converted to one or more corresponding fatty acetate esters. In certain embodiments, chemically converting one or more fatty alcohols to one or more corresponding fatty acetate esters comprises contacting one or more fatty alcohols with acetic anhydride .

[00345] A method of producing one or more fatty alcohols, one or more fatty aldehydes and / or one or more fatty acetic acids from one or more unsaturated lipid moieties disclosed herein In any one embodiment, one or more fatty alcohols, one or more fatty aldehydes and / or one or more fatty acetic acids are further concentrated by a separation procedure.

[00346] In another aspect, producing one or more unsaturated lipid moieties for use in any of the methods of producing one or more fatty alcohols and / or one or more fatty aldehydes Disclosed herein are methods of producing a recombinant microorganism capable of.

Enzymatic engineering operation
[00347] The enzymes in the recombinant microorganism can be engineered to improve one or more aspects of the substrate for product conversion. Non-limiting examples of enzymes that can be further engineered to use in the methods of the present disclosure include desaturases (eg, fatty acyl CoA desaturases or fatty acyl ACP desaturases), cytochrome-b5 reductase, elongase , Thioesterase, glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, diacylglycerol acyltransferase, lipid binding protein, glucose-6-phosphate dehydrogenase, and / or 6-phosphogluconate dehydrogenase, and combinations thereof including. These enzymes have improved catalytic activity, improved selectivity, improved stability, improved resistance to different fermentation conditions (temperature, pH etc), or different metabolic substrates, products, byproducts, intermediates It is possible to engineer for improved resistance to the body etc.

[00348] The desaturase enzyme produces E for the E product or for the Z product due to improved hydrocarbon selectivity due to the improved catalytic activity in the desaturation of unsaturated substrates It is possible to engineer for improved selectivity of objects. For example, a Z9 fatty acyl desaturase may additionally or alternatively be used to improve the yield of substrate for product conversion of saturated fatty acyl CoA to the corresponding unsaturated fatty acyl CoA. It is possible to engineer to improve the selectivity of the desaturation at position 9 to produce. In a further non-limiting example, the cytochrome b5 reductase can be engineered to improve the catalytic activity in the exchange of reducing equivalents between the one electron carrier and the two electron carrying nicotinamide dinucleotide; Elongase enzymes can be engineered to improve the catalytic activity of fatty acid substrate elongation; thioesterases for the improvement of catalytic activity in the conversion of acyl ACP or acyl CoA to free fatty acids It is possible to engineer; glycerol-3-phosphate acyltransferase to improve the catalytic activity of acylation at the sn-1 position of glycerol-3-phosphate Lysophosphatidic acid acyltransferase has a catalytic activity at the acylation of the sn-2 position of triacylglycerol It is possible to engineer to improve; diacylglycerol acyltransferase for improving the catalytic activity in the addition of the acyl group to the sn-3 position of diacylglycerol to form triacylglycerol. It is possible to engineer; lipid binding proteins can be engineered to improve sterol binding; glucose-6-phosphate dehydrogenase is of glucose-6-phosphate Engineered to alter or improve the selectivity of the enzyme to the coenzyme for improved catalytic activity in the oxidation to 6-phospho D-glucono-1,5-lactone 6-phosphogluconate dehydrogenase is a D-gluconic acid 6- to ribulose 5-phosphate. For improvement of catalytic activity in the oxidation of phosphate, additionally or alternatively, to be or improve change the selectivity of the enzyme for the coenzyme, which is engineered to be capable of operating.

[00349] The term "improved catalytic activity" as used herein with respect to a specific enzyme activity refers to a non-engineered desaturase (eg, fatty acyl CoA desaturase or fatty acyl ACP desaturase), cytochrome b5 Comparison of Reductase, Elongase, Thioesterase, Glycerol-3-Phosphate Acyltransferase, Lysophosphatidic Acid Acyltransferase, Diacylglycerol Acyltransferase, Lipid-Binding Protein, Glucose-6-Phosphate Dehydrogenase, or 6-Phosphogluconate Dehydrogenase etc A possible level of enzyme activity that is higher than that measured for a non-engineered enzyme. For example, overexpression of a particular enzyme can increase the level of intracellular activity for that enzyme. Desaturases (eg, fatty acyl CoA desaturase or fatty acyl ACP desaturase), cytochrome b5 reductase, elongase, thioesterase, glycerol 3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, diacylglycerol acyltransferase, lipid binding protein, glucose Mutations can be introduced into the 6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase to obtain an engineered enzyme with improved catalytic activity. Methods for increasing enzyme activity are known to those skilled in the art. Such techniques increase plasmid copy number and / or use of a stronger promoter and / or use of an activating riboswitch, introduction of mutations that reduce negative regulation of the enzyme, increase specific activity and / or the introduction of specific mutations that reduce the K _M for substrate or can include increasing the expression of the enzyme by directed evolution. See, for example, Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003).

Metabolic engineering-Enzyme overexpression for increased pathway Flux, and gene loss / downregulation
[00350] In various embodiments described herein, exogenous and endogenous enzymes in recombinant microorganisms involved in the biosynthetic pathways described herein can be overexpressed.

[00351] The terms "overexpressed" or "overexpression" refer to proteins in cells as compared to similar unaltered cells expressing basal levels of mRNA or having basal levels of protein ( Elevated levels of mRNA encoding (eg, abnormal levels) and / or elevated levels of protein (s). In certain embodiments, the mRNA (s) or protein (s) are at least 2-fold, 3-fold, 4 in a microorganism engineered to exhibit increased gene mRNA, protein, and / or activity. It can be overexpressed by 2, 5, 6, 8, 10, 12, 15 or more times.

[00352] In some embodiments, a recombinant microorganism of the present disclosure is produced from a host containing an enzymatic ability to synthesize substrate fatty acids. In this particular embodiment, for example, increasing the amount of fatty acid available for chemical conversion to FFA and / or FAAE and subsequent reduction to one or more fatty alcohols and / or one or more fatty aldehydes In order to increase the synthesis or accumulation of fatty acids, it may be useful.

[00353] In some embodiments, it may be useful to increase the expression of endogenous or exogenous enzyme to increase intracellular levels of coenzyme. In one embodiment, the coenzyme is NADH. In another embodiment, the coenzyme is NADPH. In one embodiment, expression of the protein in the pentose phosphate pathway is increased to increase intracellular levels of NADPH. The pentose phosphate pathway is an important catabolic pathway to supply reducing equivalents and an anabolic pathway important for biosynthetic reactions. In one embodiment, glucose-6-phosphate dehydrogenase, which converts glucose-6-phosphate to 6-phospho D-glucono-1,5-lactone, is overexpressed. In some embodiments, glucose-6-phosphate dehydrogenase is ZWF1 from yeast. In another embodiment, the glucose-6-phosphate dehydrogenase is ZWF1 (YNL241C) from Saccharomyces cerevisiae. In one embodiment, glucose-6-phosphate-1-dehydrogenase, which converts D-glucopyranose-6-phosphate to 6-phospho D-glucono-1,5-lactone, is overexpressed. In another embodiment, glucose-6-phosphate-1-dehydrogenase is zwf from bacteria. In certain embodiments, glucose-6-phosphate-1-dehydrogenase is purified from E. coli. It is zwf (NP_416366) derived from E. coli. In one embodiment, 6-phosphogluconolactonase, which converts 6-phospho D-glucono-1,5-lactone to D-gluconic acid 6-phosphate, is overexpressed. In some embodiments, the 6-phosphogluconolactonase is a yeast SOL3. In certain embodiments, the 6-phosphogluconolactonase is SOL3 (NP — 012033) of Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconolactonase is yeast SOL4. In certain embodiments, the 6-phosphogluconolactonase is SOL4 (NP_011764) of Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconolactonase is a bacterial pgl. In certain embodiments, the 6-phosphogluconolactonase is E. coli. It is pgl of E. coli (NP_415288). In one embodiment, 6-phosphogluconate dehydrogenase, which converts D-gluconic acid 6-phosphate to D-ribulose 5-phosphate, is overexpressed. In some embodiments, the 6-phosphogluconate dehydrogenase is GND1 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND1 (YHR183W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is GND2 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND2 (YGR256W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is gnd derived from bacteria. In certain embodiments, the 6-phosphogluconate dehydrogenase is It is gnd (NP_416533) derived from E. coli. In one embodiment, a transaldolase that interconverts D-glyceraldehyde 3-phosphate and D-sedheptulose 7-phosphate to β-D-fructofuranose 6-phosphate and D-erythrose 4-phosphate overexpresses Be done. In some embodiments, the transaldolase is TAL1 in yeast. In certain embodiments, the transaldolase is TAL1 (NP — 013458) of Saccharomyces cerevisiae. In some embodiments, the transaldolase is NQM1 in yeast. In certain embodiments, the transaldolase is NQM1 (NP_011557) of Saccharomyces cerevisiae. In some embodiments, the transaldolase is a bacterial tal. In certain embodiments, the transaldolase is E. coli. E. coli talB (NP_414549). In certain embodiments, the transaldolase is E. coli. E. coli talA (NP_416959). In one embodiment, D-erythrose 4-phosphate and D-xylulose 5-phosphate are interconverted to β-D fructofuranose 6-phosphate and D-glyceraldehyde 3-phosphate and / or D- Transketolase, which interconverts sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate with D-ribose 5-phosphate and D-xylulose 5-phosphate, is overexpressed. In some embodiments, the transketolase is TKL1 in yeast. In certain embodiments, the transketolase is TKL1 (NP — 015399) of Saccharomyces cerevisiae. In some embodiments, the transketolase is TKL2 in yeast. In some embodiments, the transketolase is TKL2 (NP_009675) of Saccharomyces cerevisiae. In some embodiments, the transketolase is bacterial tkt. In certain embodiments, the transketolase is E. coli. coli is tktA (YP_026188). In certain embodiments, the transketolase is E. coli. E. coli tktB (NP_416960). In one embodiment, ribose-5-phosphate ketol isomerase, which interchanges D-ribose 5-phosphate and D-ribulose 5-phosphate, is overexpressed. In some embodiments, ribose-5-phosphate ketol isomerase is yeast RKl1. In certain embodiments, the ribose-5-phosphate ketol isomerase is RK11 (NP — 014738) of Saccharomyces cerevisiae. In some embodiments, ribose-5-phosphate isomerase is a bacterial rpi. In certain embodiments, ribose-5-phosphate isomerase is E. coli. E. coli rpiA (NP_417389). In certain embodiments, ribose-5-phosphate isomerase is E. coli. E. coli rpiB (NP_418514). In one embodiment, D-ribulose-5-phosphate 3-epimerase, which interconverts D-ribulose 5-phosphate and D-xylulose 5-phosphate, is overexpressed. In some embodiments, D-ribulose-5-phosphate 3-epimerase is RPE1 of yeast. In certain embodiments, the D-ribulose-5-phosphate 3-epimerase is RPE1 (NP_012414) of Saccharomyces cerevisiae. In some embodiments, D-ribulose-5-phosphate 3-epimerase is a bacterial rpe. In one particular embodiment, D-ribulose-5-phosphate 3-epimerase is purified from E. coli. E. coli rpe (NP_417845).

[00354] In one embodiment, the expression of NADP ⁺ -dependent isocitrate dehydrogenase is increased to increase intracellular levels of coenzyme. In one embodiment, NADP ⁺ -dependent isocitrate dehydrogenase oxidizes D-threoisocitric acid to 2-oxoglutarate with concomitant formation of NADPH. In another embodiment, the NADP ⁺ -dependent isocitrate dehydrogenase oxidizes D-threoisocitric acid to 2-oxalosuccinic acid with concomitant formation of NADPH. In some embodiments, the NADP ⁺ -dependent isocitrate dehydrogenase is an IDP from yeast. In certain embodiments, the NADP ⁺ -dependent isocitrate dehydrogenase is IDP2 (YLR174W) from Saccharomyces cerevisiae. In some embodiments, the NADP ⁺ -dependent isocitrate dehydrogenase is icd from bacteria. In certain embodiments, the NADP ⁺ -dependent isocitrate dehydrogenase is E. coli. It is icd (NP — 415654) derived from E. coli.

[00355] In some embodiments, the expression of a malic enzyme that decarbonises malic acid to pyruvate and at the same time produces NADH or NADPH is increased to increase intracellular levels of coenzyme. In one embodiment, the malate enzyme is NAD ⁺ -dependent. In another embodiment, the malic enzyme is NADP ⁺ -dependent. In one embodiment, the malate enzyme is a bacterially derived NAD ⁺ -dependent malate dehydrogenase. In some embodiments, the NAD ⁺ -dependent malate dehydrogenase is It is maeA (NP — 415996) derived from E. coli. In some embodiments, the NAD ⁺ -dependent malate dehydrogenase is maeE from Lactobacillus casei (CAQ 68119). In another embodiment, the malate enzyme is a mitochondrial NAD ⁺ -dependent malate dehydrogenase from yeast. In some embodiments, the NAD ⁺ -dependent malate dehydrogenase is It is MAE1 (YKL029C) derived from S. cerevisiae. In another embodiment, the malate enzyme is a mitochondrial NAD ⁺ -dependent malate dehydrogenase from a parasitic nematode. In some embodiments, the NAD ⁺ -dependent malate dehydrogenase is M81055 from Ascaris suum. In one embodiment, the malate enzyme is a bacterially derived NADP ⁺ dependent malate dehydrogenase. In some embodiments, the NADP ⁺ -dependent malate dehydrogenase is It is maeB (NP_416958) derived from E. coli. In one embodiment, the malate enzyme is a NADP ⁺ dependent malate dehydrogenase from maize. In some embodiments, the NADP ⁺ -dependent malate dehydrogenase is me1 from Zea mays.

[00356] In some embodiments, the expression of aldehyde dehydrogenase, which oxidizes an aldehyde to a carboxylic acid while producing NADH or NADPH, is increased to increase intracellular levels of coenzyme. In one embodiment, the aldehyde dehydrogenase is NAD ⁺ -dependent. In another embodiment, the aldehyde dehydrogenase is NADP ⁺ -dependent. In one embodiment, the aldehyde dehydrogenase is a bacterial NAD ⁺ -dependent aldehyde dehydrogenase. In some embodiments, the NAD ⁺ -dependent aldehyde dehydrogenase is E. coli. It is aldA (NP_415933) derived from E. coli. In another embodiment, the aldehyde dehydrogenase is a cytosolic NADP ⁺ -dependent aldehyde dehydrogenase from yeast. In some embodiments, the NADP ⁺ -dependent aldehyde dehydrogenase is S. It is ALD6 (YPL061W) derived from S. cerevisiae. In another embodiment, the aldehyde dehydrogenase is a cytosolic NADP ⁺ -dependent aldehyde dehydrogenase from bacteria. In some embodiments, the NADP ⁺ dependent aldehyde dehydrogenase is E. coli. It is aldB (NP_418045) derived from E. coli.

[00357] In one embodiment, overexpression of the enzyme to increase intracellular levels of coenzyme comprises linking supplemental substrate supplementation to overexpression of the enzyme. In one embodiment, overexpression of the enzyme linked to complementation of the enzyme's co-substrate increases flux through the biochemical pathway. In one embodiment, the NAD ⁺ or NADP ⁺ dependent alcohol dehydrogenase is expressed with a co-substrate. In certain embodiments, alcohol dehydrogenase is expressed with an isopropanol co-substrate. In one embodiment, NAD ⁺ or NADP ⁺ dependent glucose dehydrogenase is expressed with a co-substrate. In certain embodiments, glucose dehydrogenase is expressed with a glucose co-substrate.

[00358] In one embodiment, transhydrogenase expression is increased to reciprocally exchange NADH and NADPH. In some embodiments, the transhydrogenase is a pyridine nucleotide transhydrogenase. In some embodiments, the pyridine nucleotide transhydrogenase is from bacteria. In certain embodiments, the pyridine nucleotide transhydrogenase is E. coli. pntAB derived from E. coli (beta subunit: NP_416119; alpha subunit: NP_416120). In some embodiments, the pyridine nucleotide transhydrogenase is from human. In certain embodiments, the pyridine nucleotide transhydrogenase is NNT from Homo sapiens (NP_036475). In certain embodiments, the pyridine nucleotide transhydrogenase is from Solanum tuberosum. In certain embodiments, the pyridine nucleotide transhydrogenase is from Spinacea oleracea.

[00359] Increased synthesis or accumulation can be achieved, for example, by overexpression of a nucleic acid encoding one or more of the above enzymes for the production of one or more unsaturated lipid moieties . Overexpression of the enzyme (s) useful for the production of the unsaturated lipid moiety (s) may be, for example, through increased expression of the endogenous gene (s), or exogenous gene (s) It can occur through the expression of or an increase in expression of Thus, naturally occurring organisms are easily modified to one through the overexpression of one or more nucleic acid molecules encoding one or more enzymes useful for the production of one or more unsaturated lipid moieties. Alternatively, it is possible to produce non-naturally occurring microorganisms that produce multiple unsaturated lipid moieties. In addition, it is possible to generate non-naturally occurring organisms by mutagenizing endogenous genes to increase the activity of enzymes in the pathway for the production of one or more unsaturated lipid moieties.

[00360] Given the present disclosure, one of ordinary skill in the art can readily construct the recombinant microorganisms described herein. For example, a recombinant microorganism of the present disclosure produces at least one nucleic acid encoding an enzyme of the pathway for the production of one or more unsaturated lipid moieties to produce one or more unsaturated lipid moieties. Because they can be constructed using the methods known in the art and exemplified above, exogenously expressed in sufficient amounts.

[00361] Methods for constructing non-naturally occurring hosts producing one or more unsaturated lipid moieties and testing their expression levels are, for example, by recombination and detection methods that are well known in the art. It is possible to carry out. Such methods are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubo et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, It can be seen that it is described in Md. (1999).

[00362] It is possible to express or overexpress exogenous or endogenous genes using a variety of mechanisms known in the art. For example, it encodes an enzyme that is useful in the biosynthetic pathway for producing one or more unsaturated lipid moieties exemplified herein, and is operably linked to an expression control sequence functional in the host organism It is possible to construct expression vector (s) carrying one or more nucleic acids. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and vectors and selectable sequences or markers operable for stable integration into the host chromosome. Including, including artificial chromosomes. For example, selectable marker genes can be included that provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or provide important nutrients rather than in culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators and the like that are well known in the art. If two or more exogenously encoded nucleic acids are to be coexpressed, then both nucleic acids can be inserted, for example, into a single expression vector or into separate expression vectors. In single vector expression, the encoding nucleic acid can be operably linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. . Transformation of exogenous nucleic acid sequences involved in metabolic or synthetic pathways can be ascertained using methods well known in the art.

[00363] Expression control sequences are known in the art, and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc. that provide for expression of polynucleotide sequences in host cells. Including. Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)). Exemplary expression control sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Volume 185, Academic Press, San Diego, Calif. (1990).

[00364] In various embodiments, expression control sequences can be operably linked to polynucleotide sequences. "Operably linked" means that the polynucleotide sequence and expression control sequence (s) are linked to the expression control sequence (s) when the appropriate molecule (eg, transcription activation protein) is linked. It is meant to be linked to allow gene expression. An operably linked promoter is located upstream of the selected polynucleotide sequence for the direction of transcription and translation. Operably linked enhancers can be located upstream, within or downstream of the selected polynucleotide.

[00365] In some embodiments, the recombinant microorganism is disrupted, which lacks one or more endogenous proteins of a pathway that competes with the biosynthetic pathway for production of one or more unsaturated lipid moieties. , Mutating, and / or engineered to reduce its activity.

[00366] In some embodiments, the recombinant microorganism lacks, destroys, mutates, and / or activity of one or more endogenous enzymes that catalyze the conversion of fatty acids to omega hydroxy fatty acids. It is operated to reduce the In some embodiments, the enzyme that catalyzes the conversion of fatty acid to ω-hydroxy fatty acid is BAA02210, BAA02211, BAA02212, BAA02214, AAO73952, AAO73953, AAO73955, AAO73955, AAO73958, AAO73959, AAO73960, AAO73961, AAO73957, XP_002546278, BAM 9649, AAB80867, AAB17462, ADL27534, AAU24352, AAA87602, CAA34612, ABM17701, AAA25760, CAB51047, AAC82967, is selected from the group consisting of WP_011027348 or homologs thereof.

[00367] In some embodiments, the enzyme that catalyzes the conversion of fatty acids to omega hydroxy fatty acids is monooxygenase (CYP 52). In some embodiments, the monooxygenase is selected from the group consisting of ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, ALK8, ALK9, ALK10, ALK11 and ALK12. In one particular embodiment, the monooxygenase is YALI 0 B 13 838 g, Y ALI 0 A 15 488 g, Y ALI 0 B 01 848 g, Y ALI 0 B 13 816 g, Y ALI 0 E 23 474 g, Y ALI 0 A 20 130 g, Y ALI 0 C 12 122 g, Y ALI 0 B 25 248 g, Y ALI 0 B 06 248 g, Y ALI 0 F 13 0 14 014 In certain embodiments, the monooxygenase is a Candida albicans monooxygenase selected from the group consisting of CaO19.10, CaO19.7683, CaO19.7512, CaO19.13150, CaO19.13927 and CaO19.6574. In certain embodiments, the monooxygenase is a Candida tropicalis monooxygenase selected from the group consisting of: CTRG — 03115, CTRG — 03120, CTRG — 01061, CTRG — 01060, CTRG — 02725, CTRG — 03114, and CTRG — 04959.

[00368] In some embodiments, the enzyme that catalyzes the conversion of fatty acids to omega hydroxy fatty acids is fatty alcohol oxidase. In some embodiments, the fatty alcohol oxidase is selected from the group consisting of FAO1 and FAO2. In one particular embodiment, the fatty alcohol oxidase is Yarrowia lipolytica YALI 0 B 14014 g. In certain embodiments, the fatty alcohol oxidase is a Candida albicans fatty alcohol oxidase selected from the group consisting of CaO19.61443 and CaO19.13562. In certain embodiments, the fatty alcohol oxidase is a Candida tropicalis fatty alcohol oxidase selected from the group consisting of CTRG — 03062 and CTRG — 03063.

[00369] In some embodiments, the enzyme that catalyzes the conversion of fatty acids to omega hydroxy fatty acids is a fatty alcohol dehydrogenase. In some embodiments, the fatty alcohol dehydrogenase is FADH. In certain embodiments, the fatty alcohol dehydrogenase is Yarrowia lipolytica YALI0F09603 g. In certain embodiments, the fatty alcohol dehydrogenase is a Candida albicans fatty alcohol dehydrogenase selected from the group consisting of I503_06411 and CaO 19.7600. In certain embodiments, the fatty alcohol dehydrogenase is Candida tropicalis fatty alcohol dehydrogenase CTRG — 05836.

[00370] In some embodiments, the enzyme that catalyzes the conversion of fatty acids to omega hydroxy fatty acids is an alcohol dehydrogenase. In some embodiments, the alcohol dehydrogenase is selected from the group consisting of ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and ADH8. In a particular embodiment, the alcohol dehydrogenase is a Yarrowia lipolytica alcohol dehydrogenase selected from the group consisting of YALI0E07766g, YALI0D25630g, YALI0E17787g, YALI0A16379g, YALI0A15147g, YALI0E15818g, YALI0D02167g and YALI0C12595g. In certain embodiments, the alcohol dehydrogenase is CaO19.11480, CaO19.12579, CaO19.5113, CaO19.4287, CaO19.11763, CaO19.10139, CaO19.2608, CaO19.11981, CaO19.4504, CaO19.11980, Candida albicans alcohol dehydrogenase selected from the group consisting of CaO19.12963 and CaO19.5517. In certain embodiments, the alcohol dehydrogenase is a Candida tropicalis alcohol dehydrogenase selected from the group consisting of CTRG — 06113, CTRG — 05482, CTRG — 05127 and CTRG — 05197.

[00371] In some embodiments, the enzyme that catalyzes the conversion of fatty acids to omega hydroxy fatty acids is a fatty aldehyde dehydrogenase. In some embodiments, the fatty aldehyde dehydrogenase is selected from the group consisting of FALDH1, FALDH2, FALDH3 and FALDH4. In a particular embodiment, the fatty aldehyde dehydrogenase is a Yarrowia lipolytica fatty aldehyde dehydrogenase selected from the group consisting of YALI0A17875g, YALI0E15400g, YALI0B01298g and YALI0F23793g. In one particular embodiment, the fatty aldehyde dehydrogenase is a Candida albicans fatty aldehyde dehydrogenase selected from the group consisting of CaO19.13871, CaO19.6518, CaO19.13487 and CaO19.6066. In certain embodiments, the fatty aldehyde dehydrogenase is a Candida tropicalis fatty aldehyde dehydrogenase selected from the group consisting of CTRG_05010, CTRG_04471 and CTRG_04473.

[00372] In other embodiments, the recombinant microorganism lacks, destroys, mutates, and destroys one or more endogenous enzymes that catalyze the conversion of fatty acyl CoA to α, β-enoyl CoA. And / or be manipulated to reduce its activity. In some such embodiments, the enzyme that catalyzes the conversion of fatty acyl CoA to α, β-enoyl CoA is CAA 04659, CAA 04660, CAA 04661, CAA 04662, CAA 04663, CAG 79214, AAA 34322, AAA 34 361, AAA 34 363, CAA 29901, BAA 04761, It is selected from the group consisting of AAA34891, AAB 08643, CAB 15271, BAN 55749, CAC 16516, ADK 16968, AEI 37634, WP_000973047, WP_025334422, WP_035184107, WP_026484842, CEL80920, WP_005293707, WP_005293707, WP_005883960, or their homologues.

[00373] In some embodiments, the enzyme that catalyzes the conversion of fatty acyl CoA to α, β-enoyl CoA is acyl CoA oxidase. In some embodiments, the acyl CoA oxidase is selected from the group consisting of POX1, POX2, POX3, POX4, POX5, POX6 and PXP2. In certain embodiments, the acyl CoA oxidase is Saccharomyces cerevisiae YGL205W. In certain embodiments, the acyl CoA oxidase is a Yarrowia lipolytica acyl CoA oxidase selected from the group consisting of YALI0E 32835 g, YALI 0 F 10 857 g, YALI 0 D 24750 g, YALI 0 E 27654 g, YALI 0 C 23859 g and YALI 0 E 0 565 g. In certain embodiments, the acyl CoA oxidase is a Candida albicans acyl CoA oxidase selected from the group consisting of CaO 19.1655, CaO 19.9224, CaO 19.1622, CaO 19.9221, CaO 19.5233, and CaO 19.13146. In certain embodiments, the Acyl-CoA oxidase is a Candida tropicalis Acyl-CoA oxidase selected from the group consisting of CTRG — 02374, CTRG — 02377 and CTRG — 02721. In certain embodiments, the acyl CoA oxidase is a Candida viswanathii acyl CoA oxidase selected from the group consisting of M18259, M12160 and M12161.

[00374] In some embodiments, the enzyme that catalyzes the conversion of fatty acyl CoA to α, β-enoyl CoA is a multifunctional enoyl CoA hydratase-β-hydroxyacyl CoA dehydrogenase. In some embodiments, the multifunctional enoyl CoA hydratase-β-hydroxyacyl CoA dehydrogenase is FOX2 (MFE1). In certain embodiments, the multifunctional enoyl CoA hydratase-β-hydroxyacyl CoA dehydrogenase is Saccharomyces cerevisiae YKR009C. In certain embodiments, the multifunctional enoyl CoA hydratase-β-hydroxyacyl CoA dehydrogenase is Yarrowia lipolytica YALI0E15378 g. In certain embodiments, the multifunctional enoyl CoA hydratase-β-hydroxyacyl CoA dehydrogenase is Candida albicans CaO 19.1809. In certain embodiments, the multifunctional enoyl CoA hydratase-β-hydroxyacyl CoA dehydrogenase is Candida tropicalis CTRG — 05506.

[00375] In some embodiments, the enzyme that catalyzes the conversion of fatty acyl CoA to α, β-enoyl CoA is β-ketoacyl CoA thiolase. In some embodiments, the β-ketoacyl CoA thiolase is selected from the group consisting of POT1 and FOX3. In certain embodiments, the β-ketoacyl CoA thiolase is Saccharomyces cerevisiae YIL160C. In certain embodiments, the β-ketoacyl CoA thiolase is Yarrowia lipolytica YALI0E 18568 g. In a particular embodiment, the β-ketoacyl CoA thiolase is a Candida albicans β-ketoacyl CoA thiolase selected from the group consisting of CaO19.752, CaO 19.1704 and CaO 19.9271. In certain embodiments, the β-ketoacyl CoA thiolase is a Candida tropicalis β-ketoacyl CoA thiolase selected from the group consisting of CTRG — 01068 and CTRG — 02168.

[00376] In some embodiments, the recombinant microorganism lacks, destroys, mutates, and / or reduces the activity of one or more proteins involved in peroxisome biogenesis Operated. In such embodiments, the one or more proteins involved in peroxisome biogenesis are XP_505754, XP_501986, XP_501311, XP_504845, XP_503326, XP_504029, XP_002549868, XP_002545227, XP_002547350, XP_002546990, EIW11539, EIW08094, EIW08094 It is selected from the group consisting of EIW 09743, EIW 0828 or homologs thereof.

[00377] In some embodiments, the one or more proteins involved in peroxisomal biogenesis are PEX1, PEX2, PEX3, PEX4, PEX5, PEX7, PEX8, PEX10, PEX10, PEX11, PEX12, PEX13, PEX14. , PEX15, PEX17, PEX18, PEX21, PEX22, PEX22, PEX25, PEX27, PEX28, PEX29, PEX30, PEX31, PEX31, PEX32 and PEX34. In certain embodiments, one or more proteins involved in peroxisome biogenesis are PEX2. In some embodiments, PEX2 is YJL210W from Saccharomyces cerevisiae. In some embodiments, PEX2 is YALI0_F01012g from Yarrowia lipolytica. In some embodiments, PEX2 is CaO19.11030 from Candida albicans. In some embodiments, PEX2 is CaO 19.3546 from Candida albicans. In some embodiments, PEX2 is CTRG_01657 from Candida tropicalis. In certain embodiments, the one or more proteins involved in peroxisome biogenesis are PEX12. In some embodiments, PEX12 is YMR026C from Saccharomyces cerevisiae. In some embodiments, PEX12 is YALI0_D26642 g from Yarrowia lipolytica. In some embodiments, PEX12 is CaO19.2009 from Candida albicans. In some embodiments, PEX12 is CaO 19.9560 from Candida albicans. In some embodiments, PEX12 is CTRG_01296 from Candida tropicalis. In certain embodiments, the one or more proteins involved in peroxisome biogenesis are PEX10. In some embodiments, PEX10 is YDR265W from Saccharomyces cerevisiae. In some embodiments, PEX10 is YALI0_C01023 g from Yarrowia lipolytica. In some embodiments, PEX10 is CaO19.13105 from Candida albicans. In some embodiments, PEX10 is CaO 19.5660 from Candida albicans. In some embodiments, PEX10 is CTRG_00008 from Candida tropicalis. In certain embodiments, one or more proteins involved in peroxisome biogenesis are PEX1. In some embodiments, PEX1 is YKL197C from Saccharomyces cerevisiae. In some embodiments, PEX1 is YALI0_C 15356 g from Yarrowia lipolytica. In some embodiments, PEX1 is CaO19.13818 from Candida albicans. In some embodiments, PEX1 is CaO 19.6460 from Candida albicans. In some embodiments, PEX1 is CTRG_04869 from Candida tropicalis. In certain embodiments, the one or more proteins involved in peroxisome biogenesis are PEX22. In some embodiments, PEX22 is YAL055W from Saccharomyces cerevisiae. In some embodiments, PEX22 is YALI0_F06226g from Yarrowia lipolytica. In certain embodiments, the one or more proteins involved in peroxisome biogenesis are PEX16. In some embodiments, PEX16 is YALI0_E16599g from Yarrowia lipolytica. In certain embodiments, the one or more proteins involved in peroxisome biogenesis are PEX14. In some embodiments, PEX14 is YGL153W from Saccharomyces cerevisiae. In some embodiments, PEX14 is YALI0_E09405 g from Yarrowia lipolytica. In some embodiments, PEX14 is CaO 19.1805 from Candida albicans. In some embodiments, PEX14 is CaO 19.9371 from Candida albicans. In some embodiments, PEX14 is CTRG — 00541 from Candida tropicalis.

[00378] In some embodiments, the recombinant microorganism lacks, destroys, suddenly destroys one or more endogenous proteins involved in one or more fat body storage and / or recycling pathways. Engineered to mutate and / or reduce its activity. In certain embodiments, one or more endogenous proteins are triacylglycerol lipases. In one embodiment, the triacylglycerol lipase is S. S. cerevisiae TGL3 (YMR313c). In some embodiments, the triacylglycerol lipase is YALI 0D 17534 g from Yarrowia lipolytica. In some embodiments, the triacylglycerol lipase is W5Q_03398 from Candida albicans. In some embodiments, the triacylglycerol lipase is CTRG_00057 from Candida tropicalis. In another embodiment, the triacylglycerol lipase is S. S. cerevisiae TGL4 (YKR089 c). In some embodiments, the triacylglycerol lipase is YALI 0 F 10010 g from Yarrowia lipolytica. In some embodiments, the triacylglycerol lipase is CaO 19.5426 from Candida albicans. In some embodiments, the triacylglycerol lipase is CaO19.12881 from Candida albicans. In some embodiments, the triacylglycerol lipase is CTRG_06185 from Candida tropicalis. In certain embodiments, the one or more endogenous proteins are lysophosphatidic acid acyltransferase that mediate fatty acyl transfer from a fatty acid containing triacyl glyceride. In one embodiment, lysophosphatidic acid acyltransferase is S. It is S. cerevisiae TGL5 (YOR081c). In certain embodiments, the one or more endogenous proteins are steryl ester hydrolases that mediate hydrolysis of fatty acid containing sterol esters. In one embodiment, the steryl ester hydrolase is S. It is S. cerevisiae TGL1 (YKL140w). In some embodiments, the steryl ester hydrolase is YALI0E32035 g from Yarrowia lipolytica. In some embodiments, the steryl ester hydrolase is CaO 19.2050 from Candida albicans. In some embodiments, the steryl ester hydrolase is CaO 19.9598 from Candida albicans. In some embodiments, the steryl ester hydrolase is CTRG — 01138 from Candida tropicalis. In one embodiment, the steryl ester hydrolase is S. It is S. cerevisiae YEH1 (YLL012W). In some embodiments, the steryl ester hydrolase is YALI0E00528 g from Yarrowia lipolytica. In some embodiments, the steryl ester hydrolase is CaO19.1887 from Candida albicans. In some embodiments, the steryl ester hydrolase is CaO 1 9.9443 from Candida albicans. In some embodiments, the steryl ester hydrolase is CTRG_01683 from Candida tropicalis. In one embodiment, the steryl ester hydrolase is S. S. cerevisiae YEH2 (YLR020C). In some embodiments, the steryl ester hydrolase is CTRG_04630 from Candida tropicalis. In certain embodiments, the one or more endogenous proteins are acyl CoA synthetases that reactivate fatty acyl moieties to fatty acyl CoA thioesters. In one embodiment, the acyl CoA synthetase is FAT1 (AAC17118).

[00379] In some embodiments, the recombinant microorganism catalyzes a reaction in a pathway that competes with the biosynthetic pathway for one or more unsaturated fatty acyl CoA intermediates Engineered to lack, destroy, mutate, and / or reduce the activity of the enzyme. In one embodiment, the one or more endogenous enzymes comprise one or more diacylglycerol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism lacks, destroys, mutates one or more diacylglycerol acyltransferases selected from the group consisting of YALI0E32769g, YALI0D07986g and CTRG_06209, or homologs thereof. And / or engineered to reduce its activity. In another embodiment, the one or more endogenous enzymes comprise one or more glycerol phospholipid acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism lacks, destroys, mutates one or more glycerol phospholipid acyltransferases selected from the group consisting of YALI0E16797g and CTG_04390, or homologs thereof. And / or engineered to reduce its activity. In another embodiment, the one or more endogenous enzymes comprise one or more acyl CoA / sterol acyltransferase. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism lacks, destroys, destroys, one or more acyl CoA / sterol acyltransferases selected from the group consisting of YALI0F06578g, CTRG_01764 and CTRG_01765, or homologs thereof. Engineered to mutate and / or reduce its activity.

[00380] In some embodiments, the recombinant microorganism lacks, destroys, mutates, and / or / or one or more endogenous proteins involved in one or more fatty acid elongation pathways. It is manipulated to reduce its activity. In certain embodiments, the one or more endogenous proteins are β-ketoacyl CoA synthases that catalyze the condensation of malonyl ACP and acyl CoA resulting in β-ketoacyl ACP. In one embodiment, the β-ketoacyl CoA synthase is the β-ketoacyl CoA synthase domain of yeast FAS. In certain embodiments, the one or more endogenous proteins are β-ketoacyl ACP synthase I, which condenses malonyl ACP and acyl ACP to form β-ketoacyl ACP. In one embodiment, β-ketoacyl ACP synthase I is FabB from bacteria. In certain embodiments, the one or more endogenous proteins are β-ketoacyl ACP synthase IIs that condense malonyl ACP and acyl ACP to form β-ketoacyl ACP. In one embodiment, the β-ketoacyl ACP synthase II is FabF from bacteria. In certain embodiments, the one or more endogenous proteins are β-ketoacyl ACP reductases that reduce the β-keto group of β-ketoacyl ACP with NADPH to form β-hydroxyacyl ACP. In one embodiment, the β-ketoacyl ACP reductase is FabG from bacteria. In another embodiment, the β-ketoacyl ACP reductase is a ketoreductase (KR) domain of yeast FAS. In certain embodiments, the one or more endogenous proteins are β-ketoacyl CoA reductase. In some embodiments, the β-ketoacyl CoA reductase is IFA38 from Saccharomyces cerevisiae (YBR159W). In some embodiments, the β-ketoacyl CoA reductase is YALI0A06787g from Yarrowia lipolytica. In some embodiments, the β-ketoacyl CoA reductase is CaO19.11340 from Candida albicans. In some embodiments, the β-ketoacyl CoA reductase is CaO19.3859 from Candida albicans. In some embodiments, the β-ketoacyl CoA reductase is CTRG — 00620 from Candida tropicalis. In certain embodiments, the one or more endogenous proteins are β-hydroxyacyl ACP dehydratases that dehydrate β-hydroxyacyl ACP to form trans-2-enoyl ACP. In one embodiment, the dehydratase is a β-hydroxyacyl ACP dehydratase / isomerase. In another embodiment, the β-hydroxyacyl ACP dehydratase / isomerase is FabA from bacteria. In one embodiment, the dehydratase is β-hydroxyacyl ACP dehydratase. In another embodiment, the β-hydroxyacyl ACP dehydratase is FabZ from bacteria. In another embodiment, the dehydratase is a yeast FAS dehydratase (DH) domain. In certain embodiments, the one or more endogenous proteins are β-hydroxyacyl CoA dehydratase. In some embodiments, the β-hydroxyacyl CoA dehydratase is PHS1 (TPL1) from Saccharomyces cerevisiae (YJL097W). In some embodiments, the β-hydroxyacyl CoA dehydratase is YALI 0 F 11935 g from Yarrowia lipolytica. In some embodiments, the β-hydroxyacyl CoA dehydratase is CaO 19.5156 from Candida albicans. In some embodiments, the β-hydroxyacyl CoA dehydratase is CaO19.12623 from Candida albicans. In some embodiments, the β-hydroxyacyl CoA dehydratase is CTRG_05224 from Candida tropicalis. In certain embodiments, the one or more endogenous proteins are enoyl ACP reductases that reduce trans-2-enoyl ACP to form acyl ACP. In one embodiment, the reducing enzyme is NADPH dependent trans-2-enoyl ACP reductase I. In another embodiment, the NADPH dependent trans-2-enoyl ACP reductase I is FabI from bacteria. In one embodiment, the reducing enzyme is NADPH dependent trans-2-enoyl ACP reductase II. In another embodiment, the reductase is NADPH dependent trans-2-enoyl ACP reductase II is FabK from bacteria. In one embodiment, the reducing enzyme is NADPH dependent trans-2-enoyl ACP reductase III. In another embodiment, the NADPH dependent trans-2-enoyl ACP reductase III is FabL from bacteria. In another embodiment, the reductase is an enol reductase (ER) domain of FAS in yeast. In certain embodiments, one or more endogenous proteins are enoyl CoA reductases. In some embodiments, the enoyl CoA reductase is TSC13 from Saccharomyces cerevisiae (YDL015C). In some embodiments, the enoyl CoA reductase is YALI0A04983g from Yarrowia lipolytica. In some embodiments, the enoyl CoA reductase is CaO19.10803 from Candida albicans. In some embodiments, the enoyl CoA reductase is CaO19.2933 from Candida albicans. In some embodiments, the enoyl CoA reductase is CTRG — 04406 from Candida tropicalis.

[00381] In another embodiment, the recombinant microorganism lacks, destroys, mutates, and / or reduces the activity of one or more endogenous proteins involved in the pentose phosphate pathway Operated by In some embodiments, expression of proteins in the pentose phosphate pathway is reduced to increase intracellular levels of NADH. In one embodiment, the one or more endogenous proteins are glucose-6-phosphate dehydrogenase, which converts glucose-6-phosphate to 6-phospho D-glucono-1,5-lactone. In some embodiments, glucose-6-phosphate dehydrogenase is ZWF1 from yeast. In another embodiment, the glucose-6-phosphate dehydrogenase is ZWF1 (YNL241C) from Saccharomyces cerevisiae. In one embodiment, one or more endogenous proteins are glucose-6-phosphate-1-dehydrogenase which converts D-glucopyranose-6-phosphate to 6-phospho D-glucono-1,5-lactone It is. In another embodiment, glucose-6-phosphate-1-dehydrogenase is zwf from bacteria. In certain embodiments, glucose-6-phosphate-1-dehydrogenase is purified from E. coli. It is zwf (NP_416366) derived from E. coli. In one embodiment, the one or more endogenous proteins are 6-phosphogluconolactonase, which converts 6-phospho D-glucono-1,5-lactone to D-gluconic acid 6-phosphate. In some embodiments, the 6-phosphogluconolactonase is a yeast SOL3. In certain embodiments, the 6-phosphogluconolactonase is SOL3 (NP — 012033) of Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconolactonase is yeast SOL4. In certain embodiments, the 6-phosphogluconolactonase is SOL4 (NP_011764) of Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconolactonase is a bacterial pgl. In certain embodiments, the 6-phosphogluconolactonase is E. coli. It is pgl of E. coli (NP_415288). In one embodiment, the one or more endogenous proteins are 6-phosphogluconate dehydrogenase, which converts D-gluconic acid 6-phosphate to D-ribulose 5-phosphate. In some embodiments, the 6-phosphogluconate dehydrogenase is GND1 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND1 (YHR183W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is GND2 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND2 (YGR256W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is gnd derived from bacteria. In certain embodiments, the 6-phosphogluconate dehydrogenase is It is gnd (NP_416533) derived from E. coli. In one embodiment, one or more endogenous proteins are D-glyceraldehyde 3-phosphate and D-sedheptulose 7-phosphate as β-D-fructofuranose 6-phosphate and D-erythrose 4- It is a transaldolase that interconverts to phosphate. In some embodiments, the transaldolase is TAL1 in yeast. In certain embodiments, the transaldolase is TAL1 (NP — 013458) of Saccharomyces cerevisiae. In some embodiments, the transaldolase is NQM1 in yeast. In certain embodiments, the transaldolase is NQM1 (NP_011557) of Saccharomyces cerevisiae. In some embodiments, the transaldolase is a bacterial tal. In certain embodiments, the transaldolase is E. coli. E. coli talB (NP_414549). In certain embodiments, the transaldolase is E. coli. E. coli talA (EP_416959). In one embodiment, one or more of the endogenous proteins are D-erythrose 4-phosphate and D-xylulose 5-phosphate, β-D-fructofuranose 6-phosphate and D-glyceraldehyde 3- With transketolase that interconverts to phosphoric acid and / or interconverts D-sedheptulose 7-phosphate and D-glyceraldehyde 3-phosphate to D-ribose 5-phosphate and D-xylulose 5-phosphate is there. In some embodiments, the transketolase is TKL1 in yeast. In certain embodiments, the transketolase is TKL1 (NP — 015399) of Saccharomyces cerevisiae. In some embodiments, the transketolase is TKL2 in yeast. In some embodiments, the transketolase is TKL2 (NP_009675) of Saccharomyces cerevisiae. In some embodiments, the transketolase is bacterial tkt. In certain embodiments, the transketolase is E. coli. coli is tktA (YP_026188). In certain embodiments, the transketolase is E. coli. E. coli tktB (NP_416960). In one embodiment, the one or more endogenous proteins are ribose-5-phosphate ketol isomerase which interconvert D-ribose 5-phosphate and D-ribulose 5-phosphate. In some embodiments, the ribose-5-phosphate ketol isomerase is yeast RKI1. In certain embodiments, the ribose-5-phosphate ketol isomerase is RKI1 (NP — 014738) of Saccharomyces cerevisiae. In some embodiments, ribose-5-phosphate isomerase is a bacterial rpi. In certain embodiments, ribose-5-phosphate isomerase is E. coli. E. coli rpiA (NP_417389). In certain embodiments, ribose-5-phosphate isomerase is E. coli. E. coli rpiB (NP_418514). In one embodiment, the one or more endogenous proteins are D-ribulose-5-phosphate 3-epimerase, which interconverts D-ribulose 5-phosphate and D-xylulose 5-phosphate. In some embodiments, D-ribulose-5-phosphate 3-epimerase is RPE1 of yeast. In certain embodiments, the D-ribulose-5-phosphate 3-epimerase is RPE1 (NP_012414) of Saccharomyces cerevisiae. In some embodiments, D-ribulose-5-phosphate 3-epimerase is a bacterial rpe. In one particular embodiment, D-ribulose-5-phosphate 3-epimerase is purified from E. coli. E. coli rpe (NP_417845).

[00382] In some embodiments, Y. pylori that introduces a biosynthetic pathway for the production of one or more unsaturated lipid moieties. The lipolytica microorganism is H222 ΔP ΔA ΔF ΔURA3. ΔP is Y.. 7 shows the absence of the acyl CoA oxidase gene (POX 1-6) in lipolytica. ΔA is Y.. Fig. 7 shows the absence of the (fatty) alcohol dehydrogenase gene (FADH, ADH1-7) in L. lipolytica. ΔF is Y.I. FIG. 7 shows the absence of the (fatty) alcohol oxidase gene (FAO1) in lipolytica. ΔURA3 represents the absence of the URA3 gene and yeast becomes a uracil auxotroph. Some embodiments introduce a biosynthetic pathway for the production of one or more unsaturated lipid moieties. The lipolytica microorganism is H222 ΔP ΔA ΔF. Some embodiments introduce a biosynthetic pathway for the production of one or more unsaturated lipid moieties. The lipolytica microorganism is MATA ura3-302 :: SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh6 Δadh7 Δfao1.

[00383] Yeast Y. A wild type isolate of lipolytica, preferably H222 strain, can be used as a starting strain for the construction of strains according to the present disclosure. The H222 strain was deposited at 29.04.2013 with DSMZ (Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH, D-38142 Braunschweig) under No. DSM 27 185 according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. The use of strains for further genetic processing requires a selectable marker. This selectable marker can be introduced into the strain in a manner known per se, for example in the form of uracil auxotrophs. Alternatively, it is possible to use a known uracil auxotroph strain, preferably the H222-S4 strain (Mauersberger S, Wang HJ, Gaillard in C, Barth G & Nicaud JM (2001) J Bacterial 183: 5102). -5109). The respective missing cassettes (e.g. POX 1-6, FADH, ADH 1-7, FAO1) are obtained by PCR or restriction, as described in Y. et al. transformed into Y. lipolytica H222-S4 according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, NY). It is possible to make from L. lipolytica H222 (Mauers-berger et al., (2001)). The creation of H222 ΔP ΔA ΔF ΔURA3 is described in WO 2015/086684, which is incorporated herein by reference in its entirety. Y. The lipolytica strain H222 ΔP ΔA ΔF is used as the initiating microorganism for the introduction of desaturases in the present disclosure (see, eg, Examples 3, 4 and 6).

Chemical transformation of products from naturally occurring organisms or from microbial synthesis
[00384] The present disclosure can be used to convert products obtained from one or more naturally occurring organisms or synthesized by one or more recombinant microorganisms into one or more downstream products Describe chemical conversion.

[00385] In some embodiments, the one or more unsaturated lipid moieties obtained from one or more naturally occurring organisms or produced by one or more recombinant microorganisms are It is possible to undergo chemical transformation to produce one or more fatty alcohols and / or one or more fatty aldehydes. One or more fatty alcohols and / or one or more fatty aldehydes are used as pheromone, perfume, flavor, polymer, or polymer intermediate. Non-limiting examples of chemical transformations include saponification, esterification, reduction, oxidation, metathesis and polymerization.

[00386] Unsaturated fatty carboxylic acids can be esterified by methods known in the art. For example, it is possible to convert fatty carboxylic acids to the corresponding fatty esters using a Fischer ester synthesis reaction. See, for example, Komura, K. et al., Synthesis 2008. 3407-3410.

[00387] The ester can be saponified to form carboxylate ion and alcohol by reaction with water and a base. Saponification methods include thermal saponification, cold saponification, microwave saponification and ultrasonically assisted saponification.

[00388] Carbon chain elongation can be carried out by known methods of converting unsaturated fatty alcohols into their elongated derivatives. The implementation of olefin metathesis catalysts makes it possible to increase the number of carbons on the fatty carbon chain and to give the Z or E stereochemistry to the corresponding unsaturated products.

[00389] In general, any metathesis catalyst that is stable under the reaction conditions and that does not react with functional groups on the fat substrate (eg, alcohol, ester, carboxylic acid, aldehyde, or acetate) can be used with the present disclosure . Such catalysts are, for example, the catalysts described by Grubbs (Grubbs, RH, "Synthesis of large and small molecules using olefin metathesis catalysts." PMSE Prepr., 2012), which is incorporated by reference in its entirety. Incorporated herein. Depending on the desired isomer of the olefin, a cis-selective metathesis catalyst such as, for example, Shahane et al., (Shahane, S. et al., ChemCatChem, 2013. 5 (12): 3436, which is incorporated herein by reference in its entirety. One of the catalysts described on pp. 3459) may be used. Specific catalysts 1-5 exhibiting cis selectivity are shown below (Scheme 1) (Khan, RK, et al., J. Am. Chem. Soc., 2013. 135 (28): p. 10258-. Hartung, J. et al., J. Am. Chem. Soc., 2013. 135 (28): p. 10183-5 .; Rosebrugh, LE, et al., J. Am. Chem. Soc., 2013. 135 ( 4): p. 1276-9 .; Marx, VM, et al., J. Am. Chem. Soc., 2013. 135 (1): p. 94-7 .; Herbert, MB, et al., Angew. Chem. Ed. Engl., 2013. 52 (1): p. 310-4; Keitz, BK, et al., J. Am. Chem. Soc., 2012. 134 (4): p. 2040-3. BK, et al., J. Am. Chem. Soc., 2012. 134 (1): p. 693-9; Endo, K. et al., J. Am. Chem. Soc., 2011. 133 (22): p. It has already been described in 8525-7).

[00390] Additional Z selective catalysts are described in (Cannon and Grubbs 2013; Bronner et al., 2014; Hartung et al., 2014; Pribisko et al., 2014; Quigley and Grubbs 2014), and these documents Is incorporated herein by reference in its entirety. Because of its excellent stability and functional group resistance, in some embodiments, the metathesis catalyst includes but is not limited to neutral ruthenium or osmium metal carbene complexes, and these complexes are formally +2 oxidized With the metal center being in the state, having 16 electrons, and being five-coordinated, with the general formula LL'AA'M = CRbRc or LL'AA'M = (C =) nCRbRc (Pederson and Grubbs 2002) And
M is ruthenium or osmium;
L and L ′ each independently represent an arbitrary neutral electron donor ligand, and phosphine, sulfonated phosphine, phosphite, phosphite, phosphite, arsine, anthracene, ether , An amine, an amide, an imine, a sulfoxide, a carboxyl, a nitrosyl, a pyridine, a thioether, or a heterocyclic carbene;
A and A ′ are halogen, hydrogen, C _{1 to} C ₂₀ alkyl, aryl, C _{1 to} C ₂₀ alkoxide, aryl oxide, C _{2 to} C ₂₀ alkoxycarbonyl, aryl carboxylate, C _{1 to} C ₂₀ carboxylate , An aryl sulfonyl, a C ₁ -C ₂₀ alkyl sulfonyl, a C ₁ -C ₂₀ alkyl sulfinyl independently selected anionic ligand, each ligand optionally being a C ₁ -C ₅ alkyl, halogen in _C 1 -C ₅ alkoxy; halogen or _optionally, C 1 -C ₅ alkyl, or _C 1 -C ₅ alkoxy is substituted with a phenyl group substituted with; A and A 'are both optionally May contain a bidentate ligand;
R _b and R _c are hydrogen, C _{1 to} C ₂₀ alkyl, aryl, C _{1 to} C ₂₀ carboxylate, C _{1 to} C ₂₀ alkoxy, aryloxy, C _{1 to} C ₂₀ alkoxycarbonyl, C _{1 to} C ₂₀ Each of R _b and R _c is optionally selected from alkylthio, C ₁ -C ₂₀ alkylsulfonyl and C ₁ -C ₂₀ alkylsulfinyl, each of which is optionally C ₁ -C ₅ alkyl, halogen, C ₁ -C ₅ alkoxy Or optionally substituted with a phenyl group substituted with halogen, C ₁ -C ₅ alkyl, or C ₁ -C ₅ alkoxy.

[00391] Other metathesis catalysts such as "well-defined catalysts" can also be used. Such catalysts are the shrock molybdenum metathesis catalysts described by Grubbs et al. (Tetrahedron 1998, 54: 4413-4450), 2,6-diisopropylphenylimidonepynylidenemolybdenum (VI) bis (hexafluoro-). t-butoxide) and Couturer, JL et al. (Angew. Chem. Int. Ed. Engl. 1992, 31: 628), including, but not limited to, the Base tungsten metathesis catalyst.

[00392] A catalyst that is useful in the method of the present disclosure is Peryshkov, et al., J. Am. Chem. Soc. 2011, 133: 20754-20757; Wang, et al., Angewandte Chemie, 2013, 52: 1939- 1943; Yu, et al., J. Am. Chem. Soc., 2012, 134: 2788-2799; Halford. Chem. Eng. News, 2011, 89 (45): 11; Yu, et al., Nature , 2011, 479: 88-93; Lee. Nature, 2011, 471: 452-453; Meek, et al., Nature, 2011: 471, 461-466; Flook, et al., J. Am. Chem. Soc. 2011, 133: 1784-1786; Zhao, et al., Org Lett., 2011, 13 (4): 784-787; Ondi, et al., “High activity, stabilized formulations, efficient synthesis and industrial use. of Mo- and W-based metathesis catalysts ”XiMo Technology Updates, 2015: http://www.ximo-inc.com/files/ximo/uploads/ download / Summary _3.11.15.pdf; Schrock, et al., Macromolecules, 2010: 43, 7515-7522; Peryshkov, et al., Organometallics 2013: 32, 5256-5259; Gerber, et al., Organometallics 2013: 32, 5573-5580; Marinescu, et al, Organometallics 20 12: 31, 6336 to 6343; Wang, et al., Angew. Chem. Int. Ed. 2013: 52, 1939-1943; Wang, et al., Chem. Eur. J. 2013: 19, 2726-2740. Page; and Townsend et al., J. Am. Chem. Soc. 2012: 134, 11334-11337 also included.

[00393] A catalyst that is useful in the method of the present disclosure is: WO 2014/155185; WO 2014/172534; US Patent Application Publication No. 2014/0330018; International Publication WO 2015/003815; Also includes the catalysts described in US.

[00394] Catalysts useful in the method of the present disclosure are disclosed in US Pat. No. 4,231,947; US Pat. No. 4,245,131; US Pat. No. 4,427,595; International Patent Publication WO 1991/009825; U.S. Patent 5,0877,10; U.S. Patent 5,142,073; U.S. Patent 5,146,033; International Publication WO 1992/019631; U.S. Patent No. 6,121,473; U.S. Patent No. 6,346,652; U.S. Patent No. 8,987,531; U.S. Patent Application Publication No. 2008/0119678; WO2008 / 066754; International Publication WO2009 / 094201; U.S. Patent Application Publication No. 2011/0154530; U.S. Patent Application Publication 2011 WO 2011/0077421; International Publication WO 2011/040603; International Publication WO 2011/0097642; US Patent Application Publication 2011/0238151; US Patent Application Publication 2012/0302710; International Publication WO 2012 U.S. Patent Application Publication No. 2012/0323000; U.S. Patent Application Publication No. 2013/0116434; International Publication No. WO 2013/070725; U.S. Patent Application Publication No. 2013/0274482; U.S. Patent Application Publication No. 2013/0281706 International Publication WO 2014/139679; International Publication WO 2014/169014; US Patent Application Publication No. 2014/0330018; and US Patent Application Publication No. 2014/0378637 Catalysts described also includes.

[00395] A catalyst that is useful in the method of the present disclosure is disclosed in WO 2007/075427; US Patent Application Publication No. 2007/0282148; International Publication WO 2009/126831; International Publication WO 2011/069134; Also included are the catalysts described in U.S. Patent Application Publication No. 2013/0261312; U.S. Patent Application Publication No. 2013/0296511; WO 2014/134333 and U.S. Patent Application Publication No. 2015/0018557.

[00396] Catalysts useful in the methods of the present disclosure also include those listed in the following table.

[00397] Catalysts useful in the methods of the present disclosure are disclosed in US Patent Application Publication No. 2008/0009598; US Patent Application Publication No. 2008/0207911; US Patent Application Publication No. 2008/0275247; Also included are the catalysts described in U.S. Patent Application Publication No. 2011/0282068; and U.S. Patent Application Publication No. 2015/0038723.

[00398] Catalysts useful in the method of the present disclosure are disclosed in WO 2007/140954; US Patent Application Publication No. 2008/0221345; International Publication WO 2010/037550; US Patent Application Publication No. 2010/0087644; Application Publication No. 2010/0113795; US Patent Application Publication No. 2010/0174068; International Publication WO 2011/01980; International Publication WO 2012/168183; US Patent Application Publication No. 2013/0079515; US Patent Application Publication 2013/0144060 US Patent Application Publication No. 2013/0211096; International Publication WO 2013/135776; International Publication WO 2014/001291; International Publication WO 2014/067767; US Patent Application Publication 2014/017 607 No., and a catalyst as described in U.S. Patent Application Publication No. 2015/0045558.

[00399] The catalyst is typically provided in a substoichiometric amount (eg, catalytic amount) in the reaction mixture. In certain embodiments, the amount is in the range of about 0.001 to about 50 mole% with respect to the limiting reagent of the chemical reaction, depending on which reagent is in stoichiometric excess. In some embodiments, the catalyst is present at about 40 mole% or less relative to the limiting reagent. In some embodiments, the catalyst is present at about 30 mole% or less relative to the limiting reagent. In some embodiments, the catalyst is less than about 20 mole%, less than about 10 mole%, less than about 5 mole%, less than about 2.5 mole%, less than about 1 mole%, about 0. Less than 5 mole%, less than about 0.1 mole%, less than about 0.015 mole%, less than about 0.01 mole%, less than about 0.0015 mole%, or less. In some embodiments, the catalyst is present in the range of about 2.5 mole% to about 5 mole% relative to the limiting reagent. In some embodiments, the reaction mixture contains about 0.5 mole% catalyst. If the molecular formula of the catalyst complex contains more than one metal, the amount of catalyst complex used in the reaction may be adjusted accordingly.

[00400] In some cases, the methods described herein can be performed in the absence of a solvent (eg, neat). In some cases, the method can include the use of one or more solvents. Examples of solvents that may be suitable for use in the present disclosure are benzene, p-cresol, toluene, xylene, diethyl ether, glycol, diethyl ether, petroleum ether, hexane, chlorohexane, pentane, methylene chloride, chloroform, carbon tetrachloride , Dioxane, tetrahydrofuran (THF), dimethylsulfoxide, dimethylformamide, hexamethylphosphoric acid triamide, ethyl acetate, pyridine, triethylamine, picoline and the like, and mixtures thereof, but is not limited thereto. In some embodiments, the solvent is selected from benzene, toluene, pentane, methylene chloride, and THF. In certain embodiments, the solvent is benzene.

[00401] In some embodiments, the method is performed under reduced pressure. This may be advantageous if volatile byproducts such as ethylene may be produced while the metathesis reaction is in progress. For example, removal of the ethylene byproduct from the reaction vessel can advantageously shift the equilibrium of the metathesis reaction towards the formation of the desired product. In some embodiments, the method is performed at a pressure less than about 760 Torr. In some embodiments, the method is performed at a pressure less than about 700 Torr. In some embodiments, the method is performed at a pressure less than about 650 Torr. In some embodiments, the method is performed at a pressure of less than about 600 Torr. In some embodiments, the method is performed at a pressure less than about 550 Torr. In some embodiments, the method is performed at a pressure less than about 500 Torr. In some embodiments, the method is performed at a pressure less than about 450 Torr. In some embodiments, the method is performed at a pressure less than about 400 Torr. In some embodiments, the method is performed at a pressure less than about 350 Torr. In some embodiments, the method is performed at a pressure less than about 300 Torr. In some embodiments, the method is performed at a pressure of less than about 250 Torr. In some embodiments, the method is performed at a pressure of less than about 200 Torr. In some embodiments, the method is performed at a pressure of less than about 150 Torr. In some embodiments, the method is performed at a pressure of less than about 100 Torr. In some embodiments, the method is performed at a pressure of less than about 90 Torr. In some embodiments, the method is performed at a pressure of less than about 80 Torr. In some embodiments, the method is performed at a pressure less than about 70 Torr. In some embodiments, the method is performed at a pressure of less than about 60 Torr. In some embodiments, the method is performed at a pressure of less than about 50 Torr. In some embodiments, the method is performed at a pressure of less than about 40 Torr. In some embodiments, the method is performed at a pressure less than about 30 Torr. In some embodiments, the method is performed at a pressure of less than about 20 Torr. In some embodiments, the method is performed at a pressure of about 20 torr.

[00402] In some embodiments, the method is performed at a pressure of about 19 Torr. In some embodiments, the method is performed at a pressure of about 18 Torr. In some embodiments, the method is performed at a pressure of about 17 Torr. In some embodiments, the method is performed at a pressure of about 16 Torr. In some embodiments, the method is performed at a pressure of about 15 torr. In some embodiments, the method is performed at a pressure of about 14 Torr. In some embodiments, the method is performed at a pressure of about 13 torr. In some embodiments, the method is performed at a pressure of about 12 torr. In some embodiments, the method is performed at a pressure of about 11 Torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 9 Torr. In some embodiments, the method is performed at a pressure of about 8 Torr. In some embodiments, the method is performed at a pressure of about 7 Torr. In some embodiments, the method is performed at a pressure of about 6 Torr. In some embodiments, the method is performed at a pressure of about 5 torr. In some embodiments, the method is performed at a pressure of about 4 Torr. In some embodiments, the method is performed at a pressure of about 3 torr. In some embodiments, the method is performed at a pressure of about 2 torr. In some embodiments, the method is performed at a pressure of about 1 torr. In some embodiments, the method is performed at a pressure less than about 1 Torr.

[00403] In some embodiments, the two metathesis reactants are present in equimolar amounts. In some embodiments, the two metathesis reactants are not present in equimolar amounts. In certain embodiments, the two reactants are about 20: 1, 19: 1, 18: 1, 17: 1, 16: 1, 15: 1, 14: 1, 13: 1, 12: 1, 11 : 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1 Present at a molar ratio of 1:16, 1:17, 1:18, 1:19 or 1:20. In certain embodiments, the two reactants are present in a molar ratio of about 10 to 1. In certain embodiments, the two reactants are present in a molar ratio of about 7 to 1. In certain embodiments, the two reactants are present in a molar ratio of about 5 to 1. In certain embodiments, the two reactants are present in a molar ratio of about 2 to 1. In certain embodiments, the two reactants are present in a molar ratio of about 1 to 10. In certain embodiments, the two reactants are present in a molar ratio of about 1 to 7. In certain embodiments, the two reactants are present in a molar ratio of about 1 to 5. In certain embodiments, the two reactants are present in a molar ratio of about 1 to 2.

[00404] In general, reactions with many of the metathesis catalysts disclosed herein provide better than 15%, better than 50%, better than 75%, or better than 90% yields. . Further, the reactants and products are selected to provide a boiling point difference of at least 5 ° C., greater than 20 ° C., or greater than 40 ° C. Furthermore, the use of metathesis catalysts allows for much faster product formation than by-products, and it is desirable to carry out these reactions as quickly as practically possible. In particular, the reaction is performed in less than about 24 hours, less than 12 hours, less than 8 hours, or less than 4 hours.

[00405] Those skilled in the art may appreciate that time, temperature and solvents may be dependent on one another, and methods of the present disclosure require changing one to another to prepare pyrethroid products and intermediates. Will recognize that there is a possibility. The metathesis process can proceed at various temperatures and times. Generally, reactions in the disclosed method are carried out using reaction times of minutes to days. For example, reaction times of about 12 hours to about 7 days can be used. In some embodiments, reaction times of 1 to 5 days can be used. In some embodiments, reaction times of from about 10 minutes to about 10 hours can be used. Generally, the reaction in the disclosed method is carried out at a temperature of from about 0 ° C to about 200 ° C. For example, the reaction can be carried out at 15-100 ° C. In some embodiments, the reaction can be performed at 20-80 ° C. In some embodiments, the reaction can be performed at 100-150 ° C.

[00406] The unsaturated fatty esters can be reduced using a suitable reducing agent that selectively reduces the ester to the corresponding aldehyde or alcohol but not the double bond. Unsaturated fatty esters can be reduced to the corresponding unsaturated fatty aldehydes using hydrogenated diisobutylaluminum (DIBAL) or Vitride®. Unsaturated fatty aldehydes can be reduced to the corresponding fatty alcohols, for example with DIBAL or Vitride®. In some embodiments, unsaturated fatty esters can be reduced to the corresponding fatty alcohols using AIH ₃ or 9-borabicyclo (3.3.1) nonane (9-BBN) (Galatis, P. et al. 2001. New York: John Wiley &Sons; and Carey & Sunderburg. Organic Chemistry, Part B: Reactions and Synthesis, 5th edition. 2007. New York. See Springer Sciences)).

[00407] In some embodiments, partial reduction of the ester to the aldehyde without overreduction to the alcohol is useful for the synthesis of the aldehyde. Partial reducing agents include lithium diisobutyl-t-butoxyaluminum hydride (LDBBA) (Kim MS et al. (2007) Tetrahedron Lett. 48: 5061) and sodium diisobutyl-t-butoxyaluminum hydride (SDBBA) (Song JI and An DK (2007) Chem. Lett. 36: 886). Red-Al (sodium bis [2-methoxyethoxy] aluminum hydride) derivatives of secondary amines (e.g. morpholine, N-methyl piperazine or pyrrolidine) can be used for partial reduction of esters or diesters . In certain embodiments, the reducing agent comprises a bulky cyclic nitrogen Lewis base that permits selective reduction of unsaturated fatty esters to fatty aldehydes. In some embodiments, modified Red-Al is prepared by reacting commercially available Red-Al with cis-2,6-dimethylmorpholine (Shin WK et al. (2014) Bull. Korean Chem. Soc. 35: 2169).

[00408] Reduction of the ester to the corresponding alcohol can also be accomplished through hydrogenolysis using a transition metal catalyst. Examples of homogeneous hydrocracking catalysts include, but are not limited to, the Group VIII catalysts described by Goussev et al. (US 20160023200 A1), which is incorporated herein in its entirety.

[00409] The catalysts or precatalysts disclosed by Goussev et al. Can be in the form of transition metal complexes of Formula IV or V,
M (PWNN) X _k Y (IV)
M (PWNWP) X _k Y (V)
M is a transition metal;
Each X simultaneously or independently represents hydrogen or a halogen atom, a C ₁ -C ₅ alkyl radical, a hydroxyl group, or a C ₁ -C ₇ alkoxy radical;
Y is CO, NO, carbene, isonitrile, nitrile, phosphite, phosphite, or phosphine such as PMe ₃ , PPh ₃ , PCy ₃ , P (iPr) ₃ ;
k is an integer 1 or 2;
PWNN and PWNWP are ligands represented by Formula A,

Each R ¹ , R ² , R ³ , and R ⁴ is independently H, substituted or unsubstituted linear or branched C _{1 to} C ₁₂ alkyl (C _{1 to} C ₈ alkyl or C _{8 to} C ₁₂ alkyl, etc.) A) substituted or unsubstituted cyclic C ₃ -C ₁₂ alkyl (cyclic C ₃ -C ₈ alkyl or cyclic C ₈ -C ₁₂ alkyl), substituted or unsubstituted C ₃ -C ₁₂ alkenyl (C ₃ -C ₈ alkenyl or C ₈ -C ₁₂ alkenyl), or substituted or unsubstituted aryl or heteroaryl groups, or any ^{two of} the R ² , R ³ , R ⁴ groups when taken together with the atoms to which these compounds are attached And form an optionally substituted saturated or partially saturated cycloalkyl, or an optional aryl or heteroaryl;
W is an oxygen atom or an NH group;
W 'is an oxygen or nitrogen atom;
The dashed line is present and represents the presence of one bond of a double bond or not present;
R is absent, H, substituted or unsubstituted linear or branched C _{1 to} C ₁₂ alkyl (such as C _{1 to} C ₈ alkyl or C _{8 to} C ₁₂ alkyl), substituted or unsubstituted cyclic C ₃ to C C ₁₂ alkyl (such as a cyclic _C 3 -C ₈ alkyl or cyclic _C 8 _{-C 12} alkyl), substituted or unsubstituted _C 3 _{-C 12} alkenyl _(such as C 3 -C ₈ alkenyl or _C 8 _{-C 12} alkenyl) Or a substituted or unsubstituted aryl or heteroaryl group;
R ′ is H, substituted or unsubstituted linear or branched C _{1 to} C ₁₂ alkyl (such as C _{1 to} C ₈ alkyl or C _{8 to} C ₁₂ alkyl), substituted or unsubstituted cyclic C _{3 to} C ₁₂ alkyl (such as Cyclic C ₃ -C ₈ alkyl or cyclic C ₈ -C ₁₂ alkyl, etc.), substituted or unsubstituted C ₃ -C ₁₂ alkenyl (eg C ₃ -C ₈ alkenyl or C ₈ -C ₁₂ alkenyl), or substitution or Unsubstituted aryl or heteroaryl groups, together with the atoms to which PR ₂ or R ⁵ and these compounds are attached, substituted or unsubstituted heteroaryl (in this non-limiting example are pyridyl, furanyl, imidazolyl, pyrazolyl or oxazolyl Form);
n and m are each independently an integer 1 or 2;
In the PWNN ligand, W is a nitrogen atom, in the PWNWP ligand W is an oxygen or nitrogen atom, and R ′ is PR ₂ ;
The metal complexes of formula IV or V are neutral or cationic.

[00410] Other embodiments of the catalyst disclosed by Goussev et al. (US 20160023200 A1) are also encompassed by the present application.

[00411] Another exemplary embodiment of a homogeneous hydrocracking catalyst for the reduction of an ester to the corresponding alcohol is disclosed in Saudan et al. (US Patent No. 8, 124, 816), The literature is incorporated herein in its entirety.

[00412] The ruthenium complex catalyst or precatalyst disclosed by Saudan et al. Can be in the form of ionic or neutral species. According to an embodiment of the present invention, the ruthenium complex has the general formula [Ru (L4) Y ₂ ] (1)
[Ru (L4) (X) _n (Y) _2-n ] (Z) _n (2)
Is possible,
L 4 represents a tetradentate ligand in which the coordination group consists of at least one amino or imino group and at least one phosphino group;
Each Y, simultaneously or independently, also represents CO, hydrogen or a halogen atom, a hydroxyl group, or a C _{1 to} C ₆ alkoxy or carboxylic acid radical, or a BH ₄ or ALH ₄ group;
X represents C ₃ -C ₃₀ monophosphine or solvent;
Z represents a non-coordinating anion;
n is 0, 1 or 2.

In particular, L 4 represents a tetradentate ligand, such as a C ₈ -C ₄₅ compound, whose coordinating group consists of two amino or imino and two phosphino groups, in particular the amino group is primary (ie NH ₂₎ Or secondary (i.e., NH) amino groups.

In a particular embodiment of the present invention, in formula (1) or (2) each Y may simultaneously or independently be a hydrogen or chlorine atom, a hydroxy radical, a C ₁ to C ₆ radical such as methoxy, ethoxy or isopropoxy radical. It represents a C ₁ -C ₆ acyloxy radical, such as a C ₆ alkoxy radical, or a CH ₃ COO or CH ₃ CH ₂ COO radical. More preferably, each Y simultaneously or independently represents a hydrogen or chlorine atom, a methoxy, ethoxy or isopropoxy radical, or a CH ₃ COO or CH ₃ CH ₂ COO radical.

In a particular embodiment of the invention, in formula (2), X represents a monophosphine of formula PR ^d ₃ and R ^d is an optionally substituted linear, branched or cyclic alkyl, alkoxy or aryloxy group C ₁ -C ₁₂ groups such as substituted or unsubstituted phenyl, diphenyl or naphthyl or dinaphthyl groups. In particular, R ^d may represent a substituted or unsubstituted phenyl, diphenyl or naphthyl or dinaphthyl group.

X may be a solvent and the term "solvent" should be understood according to the ordinary meaning in the art, including but not limited to the compounds used as diluents in the preparation of the complex or in the process of the present invention examples include dimethyl sulfoxide, acetonitrile, dimethylformamide, alcohols _(e.g., C 1 -C ₄ alcohol), or on THF, which is a substrate of acetone, pyridine or _C 3 -C ₈ esters or processes of the present invention.

In certain embodiments of the present invention, in Formula (2), Z represents a halogen atom, a hydroxyl group, or a C ₁ -C ₆ alkoxy, phenoxy or carboxylic acid radical.

  The complexes of formula (1) generally represent preferred embodiments of the invention for practical reasons.

[00413] An embodiment of the L4 tetradentate ligand is as described in Saudan et al. Other embodiments of the catalyst disclosed in Saudan et al. (US Pat. No. 8,124,816) are encompassed by the present application.

[00414] Fatty alcohols can be oxidized to fatty aldehydes by an oxidation process. In some embodiments, the oxidation step comprises one or more partial oxidation methods. In certain embodiments, one or more partial oxidation methods include NaOCI / TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl). The oxidation can be stopped at the aldehyde stage by carrying out the oxidation for a short time. Alternatively, the oxidation can proceed to the carboxylic acid stage by adding a phase transfer catalyst that causes a large acceleration of the oxidation. US Pat. No. 6,127,573 describes a TEMPO catalyzed oxidation of primary alcohols to carboxylic acids using CaClO ₃ and NaClO, which is incorporated herein in its entirety. Zhao et al., Organic Synthesis, Vol. 81, p. 195 (2005) describes the oxidation of primary alcohols to carboxylic acids with sodium chloride catalyzed by TEMPO and bleach.

[00415] In TEMPO oxidation, the secondary oxidant converts TEMPO, or related stable radicals, in the oxoammonium salt that functions as a primary oxidant, converting the alcohol to the corresponding aldehyde. This results in the formation of hydroxylamine which is oxidized to TEMPO radicals, thus completing the catalytic cycle.

[00416] The most common stoichiometric oxidant in TEMPO-mediated conversion of primary alcohols to aldehydes is sodium hypochlorite (NaOCl) (Anelli's oxidation, Anelli PL et al., (1987) J. Org. Chem. 52: 2559 pages); sodium chlorite _{(NaClO 2) (Zhao's modification} of Anelli's oxidation, Zhao M et al, (1999) J.Org.Chem 64:. 2564 pages); and PhI _{(OAc) 2} (Oxidation of Epp and Widlanski, Epp JB and Widlanski TS (1999) J. Org. Chem. 64: 293). Other stoichiometric oxidants used but less common are MCPBA (Cella JA et al., (1975) J. Org. Chem. 40: 1860); Ca (ClO) ₂ (swimming pool bleach) Res. 338: p. 835); t-BuOCl (Li K and Helm RF (1995) Carbohydr. Res. 273: p. 249; Rye CS and Withers SG (in: Ying L and Gervay-Hague J (2003) Carbohydr. Res. 338: p. 835); . 2002) J.Am.Chem.Soc 124: 9756 _{pp); CuCl-O 2 (Semmelhack} MF et al, (1984) J.Am.Chem.Soc 106:. 3374 pp); NaBrO ₂ (Inokuchi T et al , (1990) J.Org.Chem 55:. 462 pp); Cl ₂ (Merbouh N et al, (2002) J.Carbohydr Res 21:.. 65 pp); Br ₂ (Merbouh et al, 2002); And trichloroisocyanuric acid (De Luca L et al. (2003) J. Org. Chem. 68: 4999). It is possible to carry out the oxidation under electrochemical conditions in the presence of the catalyst TEMPO (Schnatbaum K and Schafer HJ (1999) Synthesis 5: p. 864).

[00417] In some embodiments, 4,4-dimethyloxazolidine-N-oxyl (DOXYL), 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) and 4-hydroxy-TEMPO and its Other di-t-alkyl nitroxyls such as derivatives as well as nitroxyl described in WO 95/07303 can be used instead of TEMPO.

[00418] In another embodiment, the oxidation process is copper catalyzed aerobic oxidation as described by McCann and Stahl 2015 (Mccann SD and Stahl SS (2015) Acc. Chem. Res. 48: 1756-1766). This document is incorporated herein in its entirety.

Pheromone composition and use thereof
[00419] In one aspect, it is produced by any of the methods of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties disclosed herein One or more compositions are provided.

[00420] An exemplary insect pheromone in the form of a fatty alcohol, fatty aldehyde, or fatty acetic acid that can be made using the recombinant microorganisms and methods described herein is (Z) -11- Hexadecenal, (Z) -11-hexadecenyl acetate, (Z) -9-tetradecenyl acetate, (Z, Z) -11, 13-hexadecadienal, (9 Z, 11 E) -hexadecadienal Nal, (E, E) -8, 10-dodecadien-1-ol, (7E, 9Z) -dodecadienyl acetate, (Z) -3-nonen-1-ol, (Z) -5-decene-1 -Ol, (Z) -5-decenyl acetate, (E) -5-decene-1-ol, (E) -5-decenyl acetate, (Z) -7-dodecene-1-ol, Z) -7-dodecenyl acetate, ( ) -8-Dodecene-1-ol, (E) -8-dodecenyl acetate, (Z) -8-Dodecene-1-ol, (Z) -8-dodecenyl acetate, (Z)- 9-dodecene-1-ol, (Z) -9-dodecenyl acetate, (Z) -9-tetradecen-1-ol, (Z) -11-tetradecen-1-ol, (Z) -11- Tetradecenyl acetate, (E) -11-tetradecen-1-ol, (E) -11-tetradecenyl acetate, (Z) -7-hexadecen-1-ol, (Z) -7-hexadecenal, (Z) -9-hexadecen-1-ol, (Z) -9-hexadecenal, (Z) -9-hexadecenyl acetate, (Z) -11-hexadecen-1-ol, (Z) -13- Octadecene 1-ol, and (Z) -13-octadecena Including Le not limited thereto.

[00421] As noted above, the product produced through the methods described herein is a pheromone. The pheromone prepared according to the method of the present invention can be formulated for use as an insect control composition. The pheromone composition can include a carrier and / or be contained in a dispenser. The carrier can be an inert liquid or solid, but is not limited thereto.

[00422] Examples of solid carriers are kaolin, bentonite, dolomite, calcium carbonate, talc, powdered magnesia, fuller's earth, wax, gypsum, diatomaceous earth, rubber, plastics, fillers such as kaolin, silica, silica gel, silicic acid, ata clay (Attaclay), limestone, chalk, ocher, clay, dolomite, calcium sulfate, magnesium sulfate, magnesium oxide, mineral soil such as soil synthetic material, ammonium sulfate, ammonium phosphate, ammonium nitrate, fertilizer such as thiourea and urea, cereal meal, Products of vegetable origin such as bark meal, wood flour and nut meal, cellulose powder, attapulgite, montmorillonite, mica, vermiculite, synthetic silica and synthetic silica, or compositions thereof, including but not limited to these.

[00423] Examples of liquid carriers are water; alcohols such as ethanol, butanol or glycol, and their ethers or esters such as methyl glycol acetate; ketones such as acetone, cyclohexanone, methyl ethyl ketone, methyl isobutyl ketone, or isophorone; hexane, pentane Or an alkane such as heptane; an aromatic hydrocarbon such as xylene or alkyl naphthalene; a mineral or vegetable oil; an aliphatic chlorinated hydrocarbon such as trichloroethane or methylene chloride; an aromatic chlorinated hydrocarbon such as chlorobenzene; dimethylformamide, dimethyl sulfoxide Or water soluble or strong polar solvents such as N-methyl pyrrolidone; liquid gas; beeswax, lanolin, shellac wax, carnauba wax, fruit wax Or waxes such as candelilla wax, microcrystalline, mineral waxes, ceresins, or other waxes such as montan; monoethanolamine salts, sodium sulfate, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium chloride, sodium acetate, sulfuric acid Ammonium hydrogen chloride, Ammonium chloride, Ammonium acetate, Ammonium formate, Ammonium oxalate, Ammonium carbonate, Ammonium hydrogencarbonate, Ammonium thiosulphate, Ammonium hydrogen phosphate, Ammonium dihydrogen phosphate, Ammonium hydrogen phosphate, Ammonium thiocyanate, Sulfamic acid Including, but not limited to, salts such as ammonium or ammonium carbamate and mixtures thereof. A bait or feeding stimulant can also be added to the carrier.

Synergist
[00424] In some embodiments, pheromone compositions are combined with active chemical agents to provide a synergistic effect. The synergy obtained by the method taught can be quantified according to the Colby equation (i.e. (E) = X + Y-(X ^* Y / 100)). See Colby, RS "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations", 1967 Weeds, Vol. 15, pages 20-22, which is incorporated herein by reference in its entirety. Thus, by "synergistic" is intended a component that, due to its presence, increases the desired effect above the loading level. The pheromone composition and adjuvant of the present method are capable of synergistically increasing the efficacy of the agriculturally active compound as well as the agricultural auxiliary compound.

Thus, in some embodiments, pheromone compositions can be formulated with a synergist. As used herein, the term "synergist" refers to a substance that can be used with pheromone to reduce the dose of pheromone to attract at least one insect or to enhance the efficacy of the pheromone It is The synergist may or may not be an independent attractant of insects in the absence of the pheromone.

[00426] In some embodiments, the synergist is a volatile phytochemical that attracts at least one species. The term "phytochemical" as used herein means a compound naturally occurring in a plant species. In certain embodiments, the synergist is selected from the group comprising: β-caryophyllene, isocaryophyllene, α-mufrene, inalool, Z3-hexenol / yl acetate, β-farnesene, benzaldehyde, phenylacetaldehyde, and combinations thereof It is selected.

[00427] The pheromone composition can contain the pheromone and the synergist in a mixed or otherwise combined form, or the pheromone composition contains the pheromone and the synergist independently in unmixed form May be

Insecticide
[00428] The pheromone composition can include one or more insecticides. In one embodiment, the insecticide is a chemical insecticide known to those skilled in the art. Examples of chemical insecticides are
Cyfluthrin, permethrin, cypermethrin, bifinthrin (bifinthrin), fenvalerate, flucithinate, azinphos methyl, methyl parathion, buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran, clothianidin, acephate, malathion, quinolphos (quinolphos), chloropyriphos chloropychloros Prophenophos (profenophos), bendiocarb, bifenthrin, chlorpyrifos, cyfluthrin, diazinon, dinosaur, fenpropathrin, quinoprene, insecticidal soaps or oils, neonicotinoids, diamides, avermectins and derivatives, spinosad and derivatives, azadirachtin, pyridarin and its Pyrethroid or organic, including but not limited to mixtures Containing one or more down pesticides.

[00429] In another embodiment, the insecticide is one or more biological insecticides known to those skilled in the art. Examples of biological insecticides are azadirachtin (penseed oil), a toxin derived from natural pyrethrin, Bacillus thuringiencis and Beauveria bassiana, viruses (eg CYD-XTM, CYD-X HPTM, GermstarTM ), Madex HPTM, and Spod-XTM, peptides (Spear-TTM, Spear-PTM, and Spear-CTM), including, but not limited to.

[00430] In another embodiment, the insecticide is an insecticide that targets nerves and muscles. Examples are GABAergics such as acetylcholinesterase (AChE) inhibitors such as carbamates (eg methytyl and thiodicarb) and organophosphorus (eg chlorpyrifos), cyclodienes organochlorine (eg endosulfan) and phenylpyrazoles (eg fipronil) Sodium channel modulators such as chloride channel antagonists, pyrethrins and pyrethroids (eg cypermethrin and λ-cyhalothrin), nicotinic acetylcholine receptors such as neonicotinoids (eg acetamiprid, tiacloprid, thiamethoxam) Nicotinic acetylcholine receptor (nAChR) allosteric such as (nAChR) agonists, spinosyns (eg spinose and spinetram) Modulators, chloride ion channel activators such as avermectin and milbemycin (eg abamectin, emamectin benzoate), nicotinic acetylcholine receptor (nAChR) blockers such as bensultap and cartap, voltage-dependent such as indoxacarb and metaflumizone Included are ryanodine receptor modulators such as sodium channel blockers, diamides (eg, chlorantraniliprole and flubendiamide). In another embodiment, the insecticide is an insecticide that targets respiration. Examples include chemicals that uncouple oxidative phosphorylation through the disruption of proton gradients, such as chlorfenapyr and mitochondrial complex I electron transfer inhibitors.

[00431] In another embodiment, the insecticide is an insecticide that targets the midgut. Examples include microbial disruptors of insect midgut membranes such as Bacillus thuringiensis and Bacillus sphaericus.

[00432] In another embodiment, the insecticide is an insecticide that targets growth and development. Examples are juvenile hormone mimics such as juvenile hormone analogues (eg phenoxycarb), inhibitors of chitin biosynthesis, Type 0 such as benzoylureas (eg flufenoxuron, lufenuron and novaron), and diacylhydrazines Included are ecdysone receptor agonists such as (eg, methoxyfenozide and tebufenozide).

Stabilizer
[00433] According to another embodiment of the present disclosure, the pheromone composition may include one or more additives that enhance the stability of the composition. Examples of additives include, but are not limited to, fatty acids and vegetable oils such as, for example, olive oil, soybean oil, corn oil, safflower oil, canola oil, and combinations thereof.

filler
[00434] According to another embodiment of the present disclosure, the pheromone composition may comprise one or more fillers. Examples of fillers include, but are not limited to, one or more mineral clays (eg, attapulgite). In some embodiments, the attractant composition may comprise one or more organic thickeners. Examples of such thickeners include, but are not limited to, methylcellulose, ethylcellulose, and any combination thereof.

solvent
[00435] According to another embodiment, pheromone compositions of the present disclosure can include one or more solvents. Apply by brushing, immersing, spreading with a roller, spraying or otherwise applying a liquid composition to a substrate (eg, artificial bait) to which the user wishes to have a pheromone coating If the user is to use a good liquid composition, a composition containing a solvent is desirable. In some embodiments, the solvent (s) used is selected to solubilize or substantially solubilize one or more components of the pheromone composition. Examples of solvents include, but are not limited to, water, aqueous solvents (eg, mixtures of water and ethanol), ethanol, methanol, chlorinated hydrocarbons, petroleum based solvents, terpenes, xylenes, and any combination thereof.

[00436] In some embodiments, pheromone compositions of the present disclosure include an organic solvent. Organic solvents are mainly used in emulsions, ULV formulations and, to a lesser extent, granular formulations. Mixtures of solvents may be used. In some embodiments, the present disclosure teaches the use of solvents including aliphatic paraffin oils such as kerosene or refined paraffins. In other embodiments, the present disclosure teaches the use of aromatic solvents such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons are useful as co-solvents to prevent crystallization when the formulation is emulsified in water. Alcohols may also be used as cosolvents to increase solvency.

Solubilizer
[00437] In some embodiments, pheromone compositions of the present disclosure include a solubilizer. Solubilizers are surfactants, which form micelles in water at concentrations above the critical micelle concentration. The micelle can then dissolve or solubilize the water-insoluble substance inside its hydrophobic portion. The types of surfactants commonly used for solubilization are nonionics; sorbitan monooleic acid; sorbitan monooleic acid ethoxylate; and oleic acid methyl ester.

Binder
[00438] According to another embodiment of the present disclosure, the pheromone composition may comprise one or more binding agents. A binder can be used to facilitate the association of the pheromone composition with the surface of the material to which the composition is coated. In some embodiments, a binder can be used to promote the association of additives (eg, insecticides, insect growth regulators, and the like) with pheromone compositions and / or substances on the surface. It is possible. For example, the binder can include synthetic or natural resins typically used for paints and paints. These allow the insects to bite and ingest the components of the composition (eg, insecticides, insect growth regulators, and the like) while the coated surface still maintains the structural integrity of the coating. It may be modified to be unbreakable.

[00439] Nonlimiting examples of binders are polyvinyl pyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethyl cellulose, starch, vinyl pyrrolidone / vinyl acetate copolymer and polyvinyl acetate, or compositions thereof; Lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or combinations thereof; Antifoaming agents such as silicone emulsions, long chain alcohols, phosphoric esters, acetylenic diols, fatty acids or organic fluorine compounds; Complexing agents such as salts of acetic acid (EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoric acid, or combinations thereof are included.

[00440] In some embodiments, the binder also acts as a filler and / or a thickener. Examples of such binders include, but are not limited to, one or more of shellac, acrylic resins, epoxy resins, alkyds, polyurethanes, linseed oil, cutting oil, and any combination thereof.

[00441] In some embodiments, the pheromone composition comprises a surfactant. In some embodiments, surfactants are added to the liquid agricultural composition. In another embodiment, the surfactant is added to the solid agent, in particular a solid agent designed to be diluted with carrier prior to application. Thus, in some embodiments, the pheromone composition comprises a surfactant. Surfactants may also be used alone or in conjunction with other additives such as minerals or vegetable oils as an adjuvant to the spray tank mixture to improve the biological performance of the pheromone on the target . Surfactants can be anionic, cationic or nonionic in nature and can be used as emulsifiers, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactant is a non-ionic material such as alkyl ethoxylates, linear aliphatic alcohol ethoxylates, and aliphatic amine ethoxylates. Surfactants conventionally used in formulation technology and which may be used in this formulation are disclosed in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, NJ, 1998, and in Encyclopedia of Surfactants, Vol. I-III, Chemical Publishing Co., New York, 1980-81. In some embodiments, the present disclosure includes alkyls of alkali metals, alkaline earth metals or aromatic sulfonic acids such as ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acids and fatty acids of arylsulfonic acids Ethers, lauryl ethers, fatty alcohol sulfates and ammonium salts of fatty alcohol glycol ether sulfates, sulfonated naphthalene and its derivatives with formaldehyde, naphthalene or naphthalene sulfonic acid with phenol and formaldehyde, phenol or phenol Condensate of sulfonic acid and formaldehyde, condensate of phenol and formaldehyde and sodium sulfite, polyoxyethylene octyl phenyl ether, ethoxylated isooctyl- Octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylaryl polyether alcohol, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenol, salt of phosphorylated triarylphenol ethoxylate, lauryl alcohol polyglycol ether acetate, The use of surfactants is taught, including sorbitol esters, lignin sulfite waste liquors or methylcellulose, or combinations thereof.

[00442] In some embodiments, the present disclosure includes salts of alkyl sulfates such as diethanol ammonium lauryl sulfate; alkyl aryl sulfonates such as calcium dodecyl benzene sulfonate; alkyl phenol-alkylene oxide addition such as nonylphenol-C18 ethoxylate Products; alcohol-alkylene oxide adducts such as tridecyl alcohol-C16 ethoxylate; soaps such as sodium stearate; alkyl naphthalene sulfonates such as sodium dibutyl naphthalene sulfonate; sodium di (2-ethylhexyl) sulfosuccinate Dialkyl esters of sulfosuccinate salts such as; sorbitol esters such as sorbitol oleate; quaternary amides such as lauryltrimethylammonium chloride Polyethylene glycol esters of fatty acids such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of mono and dialkyl phosphate esters; soybean oil, rapeseed / canola oil, olive oil, castor oil, sunflower seed oil, coconut oil, Vegetable oils such as corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil and the like; and other suitable surfactants, including esters of such vegetable oils, in particular methyl esters Do.

Wetting agent
[00443] In some embodiments, the pheromone composition comprises a wetting agent. A humectant is a substance that, when added to a liquid, increases the expansion or penetration of the liquid by reducing the interfacial tension between the liquid and the surface over which the liquid is spread. Wetting agents increase the rate at which the powder wets in water to form a concentrate for soluble liquids or suspension concentrates during processing and manufacture, and mixing of product and water in a spray tank or other container During, it is used for two main functions in agrochemical formulations to reduce the wetting time of infiltrating powders and to improve the penetration of water into water dispersible granules. In some embodiments, examples of wetting agents used in pheromone compositions of the present disclosure, including wettable powders, suspension concentrates, and water dispersible granular formulations, are sodium lauryl sulfate, sodium dioctyl sulfosuccinate, Alkyl phenol ethoxylates and fatty alcohol ethoxylates.

Dispersant
[00444] In some embodiments, pheromone compositions of the present disclosure include a dispersant. A dispersant is a substance that adsorbs to the surface of the particles to help preserve the dispersion of the particles and to prevent the particles from reaggregating. In some embodiments, dispersants are added to the pheromone compositions of the present disclosure to facilitate dispersion and suspension during manufacture to ensure that the particles re-disperse in water in the spray tank. In some embodiments, dispersants are used in wettable powders, suspension concentrates, and water dispersible granules. Surfactants used as dispersants have the ability to adsorb strongly to particle surfaces and provide a charged or steric barrier to particle reaggregation. In some embodiments, the most commonly used surfactants are anionic, non-ionic, or a mixture of the two types.

[00445] In some embodiments, in wettable powder formulations, the most common dispersant is sodium lignin sulfonate. In some embodiments, suspension concentrates provide very good adsorption and stabilization using polyelectrolytes such as sodium naphthalene sulfonic acid formaldehyde concentrate. In some embodiments, tristyrylphenol ethoxylated phosphate esters are also used. In some embodiments, alkyl aryl ethylene oxide concentrates, EO-PO block copolymers, and the like may be combined with the anionic material as dispersants for suspension concentrates.

Polymer surfactant
[00446] In some embodiments, pheromone compositions of the present disclosure comprise a polymeric surfactant. In some embodiments, the polymeric surfactant has a very long hydrophobic "backbone" and a large number of ethylene oxide chains that form a "comb" type surfactant 'teeth'. In some embodiments, these high molecular weight polymers can provide suspension concentrates with very good long-term stability. This is because the hydrophobic skeleton has many anchoring points on the particle surface. In some embodiments, examples of dispersants used in pheromone compositions of the present disclosure are sodium lignin sulfonate, sodium naphthalene sulfonate formaldehyde concentrate, tristyryl phenol ethoxylated phosphate ester, fatty alcohol ethoxylate Alkyl ethoxylates, EO-PO block copolymers, and graft copolymers.

emulsifier
[00447] In some embodiments, pheromone compositions of the present disclosure include an emulsifier. An emulsifier is a substance that stabilizes the suspension of droplets of one liquid phase in another liquid phase. Without the emulsifier, it is believed that the two liquids separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkylphenols with 12 or more ethylene oxide units or fatty alcohols and fatty acid soluble calcium salts of dodecyl benzene sulfonic acid. A range of hydrophilic-lipophilic balance ("HLB") values from 8 to 18 will usually give a good stable emulsion. In some embodiments, emulsion stability may be improved by the addition of small amounts of EO-PO block copolymer surfactant.

Gelling agent
[00448] In some embodiments, the pheromone composition comprises a gelling agent. Thickeners or gelling agents mainly modify the rheological or flow properties of the liquid, suspension concentrates, emulsions, and suspoemulsions to prevent separation and settling of the dispersed particles or droplets. Used in the formulation. Thickening, gelling, and anti-settling agents generally fall into two categories: water insoluble particles and water soluble polymers. It is possible to make suspension concentrate formulations using clay and silica. In some embodiments, the pheromone composition comprises one or more thickeners, including but not limited to montmorillonite, eg, bentonite; magnesium aluminum silicate; and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickeners. The most commonly used types of polysaccharides are natural extracts of seeds and seaweed or synthetic derivatives of cellulose. Some embodiments utilize xanthan and some embodiments utilize cellulose. In some embodiments, the disclosure uses guar gum; locust bean gum; carrageenam; alginic acid; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC), including but not limited to thickeners Teach. In some embodiments, the present disclosure teaches the use of other types of anti-settling agents such as modified starches, polyacrylates, polyvinyl alcohols, and polyethylene oxides. Another good anti-settling agent is xanthan gum.

Defoamer
[00449] In some embodiments, the presence of surfactant lowers the interfacial tension but may cause the water based formulation to foam during the mixing operation at manufacture and application through a spray tank. Thus, in some embodiments, an antifoam is often added during the production phase or prior to filling the bottle / spray tank to reduce the tendency to foam. In general, there are two types of defoamers: silicones and non-silicones. The silicone is usually an aqueous emulsion of dimethylpolysiloxane and the non-silicone antifoam is a water insoluble oil such as octanol, nonanol or silica. In either case, the function of the defoamer is to transfer the surfactant from the gas-liquid interface.

Preservative
[00450] In some embodiments, the pheromone composition comprises a preservative.

Additional activator
[00451] According to another embodiment of the present disclosure, the pheromone composition may comprise one or more insect feeding stimulants. Examples of insect feeding stimulants include, but are not limited to, raw cottonseed oil, fatty acid esters of phytol, fatty acid esters of geranylgeraniol, fatty acid esters of other vegetable alcohols, plant extracts, and combinations thereof.

[00452] According to another embodiment of the present disclosure, the pheromone composition may include one or more insect growth regulators ("IGRs"). IGR may be used to alter insect growth and produce malformed insects. Examples of insect growth regulators include, for example, dimilin.

[00453] According to another embodiment of the present disclosure, the attractant composition infertiles or otherwise blocks captured insects, thereby reducing the population of the next generation. May contain one or more insect infertility agents. In some situations, infertile insects survive and compete with uncaptured insects for mating mate- rials more than immediate death.

Sprayable composition
[00454] In some embodiments, the pheromone compositions disclosed herein can be formulated as a sprayable composition (ie, a sprayable pheromone composition). Aqueous solvents, such as water or mixtures of water with alcohols, glycols, ketones, or other water miscible solvents can be used in the sprayable composition. In some embodiments, the water content of such mixture is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% , At least about 80%, or at least about 90%. In some embodiments, the sprayable composition is a concentrate, ie, a concentrated suspension of pheromone and other additives (eg, waxes, stabilizers, and the like) in an aqueous solvent, It is possible to dilute to the final use concentration by addition of a solvent (eg water).

[00455] In some embodiments, waxy materials can be used as a carrier for pheromone and its regioisomer in sprayable compositions. Waxes, for example, biodegradable waxes such as beeswax, carnauba wax and the like, candelilla waxes (hydrocarbon waxes), montan waxes, shellac and similar waxes, lauric acid, palmitic acid, oleic acid or stearic acid Saturated or unsaturated fatty acids such as, fatty acid amides and esters, hydroxyl fatty acid esters such as hydroxyethyl or hydroxypropyl fatty acid esters, fatty alcohols, and low molecular weight polyesters such as polyalkylene succinate are possible.

[00456] In some embodiments, it is possible to use a stabilizer with the sprayable pheromone composition. Stabilizers can be used to control the particle size of the concentrate and / or allow the preparation of a stable suspension of the pheromone composition. In some embodiments, the stabilizing agent is selected from hydroxyl and / or ethoxylated polymers. Examples include ethylene oxide and propylene oxide polymers, starches, polyhydric alcohols including starch, maltodextrin and other soluble carbohydrates or their ethers or esters, cellulose ethers, gelatin, polyacrylic acids and their salts and partial esters and the like . In other embodiments, the stabilizing agent can comprise polyvinyl alcohol such as partially hydrolyzed polyvinyl acetate and copolymers thereof. Stabilizers may be used at levels sufficient to control particle size and / or prepare a stable suspension, for example between 0.1% and 15% of an aqueous solution.

[00457] In some embodiments, it is possible to use a binder with the sprayable pheromone composition. In some embodiments, the binder further stabilizes the dispersion and / or improves adhesion of the sprayed dispersion to the target location (eg, chewing, mock bait, plants, and the like). It is possible to act on Binders include polysaccharides such as alginic acid, cellulose derivatives (acetate, alkyl, carboxymethyl, hydroxyalkyl), starch or starch derivatives, dextrin, gum (arabia, goua, locust bean, tragacanth, carrageenan, and the like), sucrose, And the like are possible. The binder can also be a non-carbohydrate water soluble polymer such as polyvinyl pyrrolidone, or an acrylic polymer such as polyacrylic acid or polymethacrylic acid in the form of acid and / or salt, or a mixture of such polymers.

Microencapsulated pheromone
[00458] In some embodiments, the pheromone compositions disclosed herein are: Ill'lchev, AL et al., J. Econ. Entomol. 2006; 99 (6): 2048-54; and Stelinki , LL. Et al., J. Econ. Entomol. 2007; 100 (4): 1360-9. It can be formulated as a microencapsulated pheromone. Microencapsulated pheromone (MEC) refers to droplets of pheromone encapsulated in a polymer capsule. The capsules control the release rate of pheromone into the surrounding environment and are small enough to be applied in the same way as used in spray insecticides. The effective field life of microencapsulated pheromone formulations can range from a few days to slightly over a week, depending on, inter alia, the climatic conditions, capsule size and chemical properties.

Sustained release preparation
[00459] The pheromone composition can be formulated to provide sustained release into the atmosphere and / or to be protected from subsequent degradation. For example, the pheromone composition can be included in a carrier such as microcapsules, biodegradable flakes and a matrix based on paraffin wax. Alternatively, pheromone compositions can be formulated as slow release sprayables.

[00460] In certain embodiments, the pheromone composition may include one or more polymeric agents known to one of ordinary skill in the art. Polymeric agents can control the rate of release of the composition into the environment. In some embodiments, the polymeric attractant composition is not affected by environmental conditions. The polymeric agent may be a sustained release agent that releases the composition in an environmentally sustained manner.

[00461] Examples of the polymer agent include cellulose, protein such as casein, fluorocarbon-based polymer, hydrogenated rosin, lignin, melamine, polyurethane, vinyl polymer such as polyvinyl acetate (PVAC), polycarbonate, polyvinylidene dinitrile Including, but not limited to, polyamide, polyvinyl alcohol (PVA), polyamidoaldehyde, polyvinyl aldehyde, polyester, polyvinyl chloride (PVC), polyethylene, polystyrene, polyvinylidene, silicone, and combinations thereof. Examples of cellulose include, but are not limited to, methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate butyrate, cellulose acetate propionate, cellulose propionate, and combinations thereof.

[00462] Other agents that can be used in sustained release or sustained release formulations include fatty acid esters (such as sebacic acid, lauric acid, palmitic acid, stearic acid or arachidic acid esters) or fatty alcohols (undecanol, Dodecanol, tridecanol, tridecenol, tetradecanol, tetradecenol, tetradecadienol (tetradecadienol), pentadecanol, pentadecenol, hexadecanol, hexadecenol, hexadecenol, hexadecadienol, octadecenol and octadecadienol including.

[00463] Using the pheromone prepared according to the methods of the present invention, as well as compositions containing pheromone, it is possible to control insect behavior and / or growth in a variety of environments. Pheromones can be used, for example, to attract or repel male or female insects to or from specific target areas. Pheromones can be used to pull insects away from vulnerable crop areas. Pheromones can be used, for example, to attract insects as part of a strategy for insect surveillance, mass capture, simulated bait / attractive insecticidal or communicative perturbation.

Simulated bait
[00464] The pheromone compositions of the present disclosure may be coated or sprayed onto the simulated bait, or the simulated bait may be otherwise impregnated with the pheromone composition.

trap
[00465] The pheromone compositions of the present disclosure may be used with any insect species, for example, moths such as moths commonly used to attract lepidopteran insects. Such bushes are well known to those skilled in the art and are commonly used in insect eradication programs in many states and countries. In one embodiment, the weir comprises one or more septums, containers, or storage containers for holding the pheromone composition. Thus, in some embodiments, the present disclosure provides a cocoon filled with at least one pheromone composition. Thus, use of the pheromone compositions of the present disclosure in a cage can result, for example, in insect surveillance, mass capture, mating disruption, or artificial bait by, for example, incorporating toxic substances into the cage to kill the captured insects. It is possible to attract insects as part of a strategy for attractant killing.

[00466] Mass capture involves placing straw at high density in the crop being protected so that a high percentage of insects are removed before the crop is damaged. The simulated bait / attractive insecticidal technique is similar except that the insects are attracted and then exposed to the killing agent. If the kill agent is an insecticide, the dispenser may also contain a chew's bait or feeding stimulant that attracts the insect and feeds an effective amount of the insecticide. The insecticide may be an insecticide known to those skilled in the art. The insecticide may be mixed with the attractant composition or may be present separately in the cage. The mixture may perform the dual function of attracting and killing insects.

[00467] Such traps can be in any suitable form, killing is not necessarily required to incorporate toxic substances, and insects are optionally killed by other means such as moribund or electrocution . Alternatively, it is possible to contaminate the insects with a fungus or virus which later kills the insects. Even if the insect is not killed, it can serve to remove male insects from areas of female insects to prevent reproduction.

[00468] Those skilled in the art will recognize that a variety of different traps are possible. Suitable examples of such weirs include water traps, adhesive traps, and one-way traps. There are many types of sticky traps. An example of an adhesive trap is a corrugated board construction, having a triangular or wedge-shaped cross section, the inner surface of which is coated with a non-drying adhesive material. Insects are trapped in contact with sticky surfaces. The water trap contains a dish of water and detergent used to catch insects. The detergent destroys the surface tension of the water and causes the insects attracted to the dish to drown in the water. One-way traps allow insects to enter the cage but prevent them from exiting. The barbs of the present disclosure can be painted bright colors to further attract insects.

[00469] In some embodiments, pheromone traps containing the composition may be combined with other types of trapping mechanisms. For example, in addition to the pheromone composition, the cocoon may include one or more flowering lamps for attracting cocoons, one or more adhesive substrates, and / or one or more colored surfaces. In other embodiments, the pheromone trap containing the composition may be free of other types of trapping mechanisms.

[00470] The fence may be placed in the field at any time of year. A person skilled in the art can easily determine the appropriate amount of composition to be used in a particular straw, and it is also possible to determine the appropriate density of straw per acre of farmland to be protected.

[00471] The chicks can be placed where insects infest (or potentially infested). Generally, moss is placed in contact with or near trees or plants. The smell of pheromone attracts insects to the cocoon. Next, the insects are captured, immobile in the cage and / or killed, for example, by the disinfectant present in the cage.

[00472] Persimmons may be placed inside orchards to overwhelm the pheromone the female emits so that the male can not just locate the female. In this regard, it is only necessary that the weir be a simple device, for example, a protected core that delivers pheromone.

[00473] The soot of the present disclosure may be provided in a ready-made form, in which case the compounds of the present disclosure have already been applied. In such instances, depending on the half life of the compound, the compound may be exposed or may be sealed in a conventional manner, as is standard with other fragrance dispensers, and the seal may Only after being placed in the position of.

[00474] Alternatively, the cocoons are sold separately, and the compounds of the present disclosure are provided in a dispensable manner such that a quantity is applied to the cocoon after the cocoon is in place. It is also good. Thus, the present disclosure may provide the compounds in a sachet or other dispenser.

dispenser
[00475] The pheromone composition can be used in conjunction with a dispenser for release of the composition in a specific environment. Any suitable dispenser known in the art can be used. Examples of such dispensers are aerosol emitters, manual dispensers, permeable barriers through which the pheromone is slowly released, rubber impregnated with the pheromone composition, plastic, leather, cotton, raw cotton, wood or wood Products include but are not limited to pads made of products, beads, tube rods, bubble caps with reservoirs with spirals or spherical ones. For example, polyvinyl chloride laminates, pellets, granules, ropes or spirals or rubber septa from which the pheromone composition evaporates. The person skilled in the art can select a suitable carrier and / or dispenser for the desired application, storage, transport or handling manner.

[00476] In another embodiment, an apparatus may be used to contaminate a male insect with a powder containing the pheromone substance itself. The polluted males then fly away and provide a source of communication disturbance by spreading the pheromone substance into the atmosphere, or by attracting other males to the polluted male rather than the real female.

Behavioral change
[00477] Using the pheromone compositions prepared according to the methods disclosed herein, it is possible to control or modulate insect behavior. In some embodiments, among other things, changing the ratio of pheromone to regioisomer in the composition such that the insects are attracted to a specific location but not in contact with the location or the insect is actually in contact with the location. By doing so, it is possible to modulate the behavior of the target insect in a modifiable manner. Thus, in some embodiments, pheromone can be used to pull insects away from the area of crops that are susceptible to attack. Thus, the present disclosure also provides a method for attracting insects to a place. The method includes providing the site with an effective amount of a pheromone composition.

[00478] The method of communication disruption may include periodically monitoring the total number or amount of insects captured. Monitoring can be done, for example, by counting the number of insects that have been supplemented for a predetermined period, such as daily, weekly, biweekly, monthly, every three months, or any other period of time selected by the observer. You may implement. Such monitoring of the supplemented insects helps to estimate the insect population during that particular time period, whereby specific types and / or doses of control in the integrated harmful species management system It can help to make decisions. For example, high insect populations may be found that may require the use of methods for the removal of insects. The early warning of new habitat visits allows for action to be taken before the population becomes unmanageable. On the contrary, if the number of insects to be discovered is small, it can be decided that it is sufficient to keep monitoring the population. The population of insects can be regularly monitored to be controlled only when it reaches a certain threshold. This provides cost effective control of insects and reduces the environmental impact of pesticide use.

Communication disturbance
[00479] It is also possible to disrupt mating using the pheromone prepared according to the methods of the present disclosure. Communication disruption disrupts the insect, disrupts the copulation's whereabouts / courtship behavior, thereby introducing an artificial stimulus (eg, the pheromone composition disclosed herein) that interferes with mating and blocks the reproductive cycle. Pest management techniques designed to control pests.

[00480] In many insect species of interest for agriculture, such as lepidopteran species, females give off an aerial odor of a specific chemical blend that constitutes the sex pheromone of that species. This airborne odor is called a pheromone plume. Such males use the information contained in the pheromone plume to locate the smelling females (known as "calling" females). Communication disruption exploits the natural response of male insects to chase plumes by introducing synthetic pheromone into insect habitats, which are designed to mimic the sex pheromone produced by female insects. Thus, in some embodiments, the synthetic pheromone utilized in communication disruption is a synthetically derived pheromone composition comprising a sex pheromone not generated by the target insect and a pheromone having a chemical structure of its regioisomer. .

[00481] The general effect of communication disruption is to disrupt male insects by shielding the natural pheromone plume and cause males to chase off "false pheromone odor marks" at the expense of finding mating partners. "Calling" is affecting the male's ability to respond to females. As a result, the male population experiences a reduced probability of successfully locating and mating females, so that breeding eventually ceases and insect attack collapses.

[00482] Communication disruption strategies include confusion, odor masking and false odor tracking. When insects are constantly exposed to high concentrations of pheromone, male insects may become unresponsive to the normal levels of pheromone released by female insects. Odor masking uses pheromones to destroy the pheromonal odors released by females. False scent tracking is performed by placing a large number of spots of pheromone at high concentrations and presenting many false scents to be tracked to the male. When released in sufficient quantities, male insects can not find the natural source of sex pheromone (female insects) and mating can not occur.

[00483] In some embodiments, the wick or moth may be adapted to release pheromone for a period corresponding at least to the breeding season (s) of the small insect to cause communicative disturbance. If the small insects have a long breeding season, or repeated breeding seasons, the disclosure may release the pheromone for a period of time, in particular about 2 weeks, generally about 1 to 4 weeks, up to 6 weeks. A can or wick may be provided which may be alternated or replaced with a subsequent similar wick. Multiple pods containing the pheromone composition may be placed, for example, in a location adjacent to the crop field. The position of the weir and the height of the weir from the ground can be selected according to methods known to those skilled in the art.

[00484] Alternatively, the pheromone composition may be dispensed from formulations such as microcapsules or twist-ties, as commonly used to disrupt pest mating.

Attractive insecticidal
[00485] The induced insecticidal method uses attractants such as sex pheromone to attract target insects to insecticidal chemicals, surfaces, devices, etc. for mass killing and ultimate population control, and captures them in large quantities It is possible to have the same effect as For example, when using a synthetic female pheromone to attract male pests, such as chicks, in an attractive insecticidal strategy, large numbers of male chicks are killed for extended periods of time to reduce mating and reproduction, and ultimately to reduce pest populations It must be suppressed. An attractant approach may be a valuable alternative to mass capture. This is because there is no need for firewood repairs or other frequent maintenance. In various embodiments described herein, the recombinant microorganism co-expresses (i) a pathway for the production of an insect pheromone and (ii) an insect harmful protein, peptide, oligonucleotide or small molecule. It is possible. In this way, the recombinant microorganism is capable of co-producing substances suitable for use in the induced insecticidal approach.

[00486] As will be apparent to those skilled in the art, the amount of pheromone or pheromone composition used for a particular application is the type and level of attack; type of composition used; concentration of active ingredient; The manner of delivery of the composition, eg, the type of dispenser used, the type of location to be handled; several factors such as the time period the method will be used; and temperature, wind speed and direction, rainfall And can vary depending on environmental factors such as humidity. One of ordinary skill in the art will be able to determine the effective amount of pheromone or pheromone composition for use in a given application.

[00487] As used herein, "effective amount" means an amount of the disclosed pheromone composition that is sufficient to affect the desired result. An effective amount can be given in one or more administrations. For example, an effective amount of composition may refer to an amount of pheromone composition sufficient to attract a given insect to a given location. Furthermore, the effective amount of the composition may refer to the amount of pheromone composition sufficient to disrupt the mating of the particular insect population of interest at a given location.

Example 1 Sourcing of Fatty Acid Precursors from Natural Sources
[00488] Reduction of fatty acids by cells expressing fatty acyl CoA reductase (FAR) is a synthetic alternative to the biohydroxylated synthesis of the proposed insect pheromone. See FIG. The reduction of fatty acids to alcohols is expected to be much easier than terminal hydroxylation. That is because (i) much more FAR sequences are known, which is a natural reaction of these enzymes, and (ii) heterogeneously catalyzed hydrogenation of acids to alcohols is well established. The ability to source target fatty acids with appropriate unsaturated groups is a major challenge to this approach.

[00489] Seed oil fatty acid database (sofa.mri.bund.de), microalgae from culture collection of Gottingen University (SAG), to understand the possibility of procuring fatty acid precursors of target insect pheromone from natural sources Reported fatty acid profiles of the strain (Lang IK et al. (2011) Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection. BMC Plant Biol. 11: 124) and general We investigated the literature.

[00490] Fourteen fatty acids that could be used as precursors for insect pheromone were investigated (see Table 4). Of these fourteen fatty acids, it has been reported that only three of Z-11-octadecenoic acid, Z-13-octadecenoic acid, and Z-11-hexadecenoic acid are found in seed oil. The plant producing the highest purity of each of these fatty acids is Asclepias Syriaca: Z-11-octadecenoic acid (44.9%) (Kuemmel DF, Chapman LR (1968) 9-hexadecenoic and 11-octadecenoic acid content of natural Lipids 3: 313-316); Cardwellia sublimis: Z-13-octadecenoic acid (22%) (Vickery JR (1971) The fatty acid composition of the seed oils of proteaceae: A chemotanomic study. Phytochemistry 10: 123 -130). Plants comprising Z-11-hexadecenoic acid are Kermadecia sinuata (40.3%) (Vickery 1971); Gevuina avellana (25.4%) (B. San, AN Yildirim, J Food Compos. Anal. 2010, 23, 706-710); Ziziphus jujube var. (B. San, AN Yildirim, J. Food Compos. Anal. 2010, 23, 706-710); Orites revoluta (35.6%) (G. Hill, RS, and Jordan, Pap. Proc. R. Soc. Tasmania 1996, 130, 9-15); Grevillea exul var. rubiginosa (45.6%) (I. Bombarda, C. Zongo, CR McGill, P. Doumenq, B. Fogliani, J. Am. Oil Chem. Soc. 2010, 87, 981-986, and M. Kato, A. Kawakita , Am J. Bot. 2004, 91, 1814-1827). Similarly for microalgae, only two of the 14 fatty acids are Z-11-octadecenoic acid (68%) produced by Pediastrum simplex and Z-11-hexadecenoic acid (35%) produced by Synechococcus elongatus in the database It is identified by. In general literature searches, 2E, 4Z, 7Z-decatrienoic acid was identified from the juice of Noni fruits (Basar S, and Westendor J (2011) Identification of (2E, 4Z, 7Z, 7Z-decatrienoic acid in Noni fruit and its Food Anal. Meth. 4: 57-65), isolated from Streptomyces viridochromogenes Tu 6 105 (7.7 mg / L) (Maier A et al., (1999) (2E, 4Z)- decadienoic acid and (2E, 4Z, 7Z) -decatrienoic acid, two herbicidal metabolites from Streptomyces viridochromogenes Tu 6105. Pestic. Sci. 55: 733-739).

[00491]

[00492] In order to determine the fatty acids that can be procured from the seed oil produced as a commodity, the literature was searched to identify the seed oil of the commodity and its composition. The main seed oils produced between 1976 and 2000 and expected for 2006 and 2020 are summarized in FIG. The main seed oils are soybeans, cottonseed, peanuts, sunflowers, canola, sesame seeds, corn, olives, palms, coconut kernels, coconuts, linseed, castor and tallow. The fatty acid composition of these oils is summarized in Table 5. The main components of general commodity seed oil are lauric acid (12: 0): coconut (47.8%) and coconut kernel (48.4%), palmitic acid (16: 0): coconut (44%) and cotton (24.7%) ), Oleic acid (18: 1Δ9c): canola (64.1%), peanuts (46.5%), coconut (39.2%), olives (76.9%) and sesame seeds (40.6%), linoleic acid (18: 2Δ9c, Δ12c): Soybean (53.2%), cotton (53.3%), sunflower (68.2%), peanuts (31.4%), and sesame (42.6%), and linolenic acid (18: 3Δ9c, Δ12c, Δ15c): flaxseed (47.4%) is there. In addition to the basic crop, modified crops are engineered to increase the content of certain fatty acids. Examples of modified soybean and canola crops are summarized in Table 6. Among these crops, if a pheromone from oleic acid can be identified, production of oleic acid with high purity over 75% would be the most promising feedstock.

[00493] In addition to the fatty acids found in the main commodity commodity seed oils, by-products with fatty acid compositions of interest are summarized in Table 7. These by-products are unique monoenes, 20: Δ5 c, 22: Δ 9 c, 18: 1 Δ 6 c, 16: 1 Δ 9 c; trienes, 18: 3 Δ 8 t, Δ 10 t, Δ 12 c, 18: 3 Δ 9 c, Δ 11 t, Δ 13 t; and trienes, 18: 3 Δ 9 , Δ11, Δ13, 4-oxo, 18: 1 Δ9 c, Δ12-hydroxy.

[00494]

[00496]

[00497] The following fatty acids are produced by plants and microalgae. Z-11-octadecenoic acid: Asclepias Syriaca (44.9%), Pediastrum simplex (68%); Z-13 octadecenoic acid: Cardwellia sublimis (22%); and Z-11-hexadecenoic acid: Kermadecia sinuata (40.3%), Synechococcus sp. elongatus (35%). The main ingredients of general commodity seed oil are lauric acid (12: 0): coconut (47.8%) and coconut kernel (48.4%); palmitic acid (16: 0): coconut (44%) and cotton (24.7%) Oleic acid (18: 1Δ9c): canola (64.1%), peanuts (46.5%), coconut (39.2%), olives (76.9%) and sesame seeds (40.6%); linoleic acid (18: 2Δ9c, Δ12c): Soybean (53.2%), cotton (53.3%), sunflower (68.2%), peanuts (31.4%) and sesame seeds (42.6%); and linolenic acid (18: 3Δ9c, Δ12c, Δ15c): flaxseed (47.4%) . Seed oils for general products are mostly composed of saturated fatty acids and monoenes.

[00498] By-products (calendula, meadowfoam, ginseng, caraway, carrots, coriander, macadamia, nutmeg, oiti sica, butternut, and castor) have unique monoenes 20: Δ5 c, 22: Δ9 c, 18: 1 Δ6 c, 16: 1Δ9c, triene 18: 3Δ8t, Δ10t, Δ12c, 18: 3Δ9c, Δ11t, Δ13t and triene oxide 18: 3Δ9, Δ11, Δ13, 4-oxo, 18: 1 Δ9c, Δ12-hydroxy.

Example 2: Background and rationale for the production of (Z) -hexadeca-11-enal by semireduction of a naturally occurring oil
[00499] Although the target pheromone is very similar to the naturally occurring fatty acids, the use of naturally occurring fatty acids as a substrate for the production of target pheromone has not been defined. In order to determine whether a vegetable oil containing a sufficient amount of (Z) -hexadeca-11-enoic acid can be used for this purpose, more information about the particular plant on the substrate side was needed.

[00500] Chilli hazelnut oil, which is the only commercial vegetable oil containing significant amounts of (Z) -hexadeca-11-enoic acid, was used as a feedstock for the production of (Z) -hexadeca-11-enal .

[00501] A schematic illustrating the overall reaction scheme is shown in FIG. After isolating the lipids from natural sources, they are subjected to transesterification. Preparation of methyl esters is a standard procedure for the production of biodiesel. The reaction may be acid catalyzed or base catalyzed. Alkaline conditions were preferred for this approach as the double bond is prone to isomerization under acidic conditions (Li H-Y et al. (2005) Chem. Phys. Lett. 404: 384-388).

[00502] Depending on the source, the resulting mixture contains only a fraction of the target material. Purification or at least enrichment of the precursor acid may be preferred to minimize the reduction equivalents required for the subsequent reduction reaction. Among the viable options are, for example, distillation and simulated moving bed (SMB) chromatography. Since the task of preliminary purification is another task, small-scale distillation was performed to determine the high temperature stability of the target compound. In the next step, purified methyl (Z) -hexadeca-11-enoate is subjected to reduction to the corresponding aldehyde. A possible reagent for this purpose would be Vitride hydrogenated (sodium bis (2-methoxyethoxy) aluminum) (Abe, T et al. (2001) Tetrahedron 57: 2701-2710). Vitride is an aluminum hydride reducing agent having the same reaction profile as DIBAL (diisobutylaluminum hydride). Unlike DIBAL, Vitride is not pyrophoric when it is exposed to air, and it reacts more gently with water, so it is safer and easier to handle. Comparing Vitride with other partial reductants known in the literature, this reductant can be used at 0 ° C while it requires -78 ° C, which is higher for similar substrates It shows selectivity and very few superreduction byproducts (Shin WK et al., (2014) Bull. Korean Chem. Soc. 35: 2169-2171).

Outline of approach
[00503] Various reported plants containing high content (> 20%) of (Z) -hexadeca-11-enoic acid were compared. Relevant parameters were oil content, cultivation conditions, overall yield and market availability.

[00504] A representative vegetable oil sample (Gevuina avellana-chili hazel) was purchased and analyzed.

[00505] A base catalyzed transesterification reaction was performed to produce methyl esters of vegetable oil fatty acids (biodiesel production).

[00506] Small scale purification / enrichment of methyl (Z) -hexadeca-11-enoate was achieved by distillation.

[00507] The semireduction of methyl ester was carried out using a modified variant of Vitride (2,6-dimethylmorpholine modified sodium bis [2-methoxyethoxy] aluminum hydride) to produce (Z) -hexadeca-11-enal .

result
[00508] The research focused on the general feasibility of sourcing natural oil and its reduction to pheromone. As the main target is (Z) -hexadeca-11-enal, this compound was the focus of investigation. The databases of SOFA (Seed Oil Fatty Acids) and SAG (Culture Collection of Algae at Goettingen University) were re-examined (see Example 1).

[00509] Part I of the report focused on the identification of plants with high content of unusual fatty acids suitable as precursors for the production of pheromone. Part II shows the proof of principle for the use of oil to produce pheromone. Part I confirms the previous search for candidate plants. A review of the whole technology seemed to be relevant, as other lipid sources such as microbial lipids are processed by exactly the same procedure. The development route of the intermediate production platform is shown in FIG.

1.1 Details of Vegetable Oil Procurement
[00510] Plants containing high content of (Z) -hexadeca-11-enoic acid have been cited in many studies examining unusual fatty acid profiles of vegetable oils (Aitzetmuller K, J. Am. Oil Chem. Soc. 81: 721-723). Interestingly, with the exception of Ziziphus jujube belonging to the Rhamnaceae family, all other plants which contain more than 20% of the (Z) -hexadeca-11-enoic acid fraction belong to the Proteaceae family.

[00511] There is very little information on oil yield and cultivation conditions. However, other parameters can be used to assess plant potential. The study concentrated on two plants. First, Grevillea exul var. Rubiginosa has 45.6% in seed oil, the largest amount of (Z) -hexadeca-11-enoic acid among all plants ever reported. Depending on the report, G. exul is a shrub or shrub up to 30 feet high and is unique to New Caledonia only. G. Further information is obtained only for other closely related species, such as robusta and generally Grevillea. Common cold resistant zones are 9 to 12, suitable for example for South California. It resists drought and deer damage, is fast growing, but is susceptible to fertilizers. No information was obtained on productivity or oil yield. Many Grevillea species are used as food plants by Lepidoptera species, such as Doli Andorra.

[00512] The second plant of interest is chili hazel. It is because this vegetable oil naturally contains about 25% (Z) -hexadeca-11-enoic acid and is commercially available. This plant is not widely grown.

1.2 Comparison of some vegetable oils by GC-FID
[00513] Chilli hazelnut oil is used in many lotions because of its sun protection properties. In many cases, different positional isomers of fatty acids are not distinguished by retailers. Chile hazelnut oil and European hazelnut oil were compared. The results are summarized in Table 8 and overlays of different GC-FIDs are shown in FIG. The only oil comprising (Z) -hexadeca-11-enoic acid is chili hazelnut oil. The 22.5% fraction is close to the values of about 22.7% and about 23.83% reported in the literature (Bertoli C et al., J. Am. Oil Chem. Soc. 75: 1037-1040; Medel F, Carillo T in Acta Hortic., International Society for Horticultural Science (ISHS), Leuven, Belgium, 2005, pp. 631-638). Although not fully studied for other sources, it is unlikely that other commercial oils contain high (Z) -hexadeca-11-enoic acid as well. It is not surprising that the European hazel (Corylus avellana) is completely different. This is because it is not associated with Chile hazel (Gevuina avellana). Surprisingly, macadamia nut oil showed only 0.59% palmitoleic acid. Usually higher contents of 16 to 34% are observed. On the other hand, the content of oleic acid was somewhat high. This is usually found to be 41-59%. There is also usually about 3% Z11-C18: 1 (Kaijser A et al. (2000) Food Chem. 71: 67-70). None of these parameters are in agreement with the reported data, leading to the conclusion that this oil is not pure macadamia nut oil.

[00514]

2. Transesterification and purification of (Z) -hexadeca-11-enoic acid
[00515] The production route considered in this study is the direct production of aldehydes by half reduction of the corresponding fatty acid derivative. In contrast to total reduction, which can also start from free fatty acids, the semireduction requires the methyl ester. Methyl esters generally have a low boiling point of about 20 K, which facilitates purification by distillation. Thus, the route that was attempted to be achieved with this approach utilized transesterification instead of saponification and distillation instead of chromatography.

2.1 Preparation of methyl (Z) -hexadeca-11-enoate / chili hazelnut biodiesel
[00516] Using the optimization protocol of Ullah et al. (2013) for the production of biodiesel from linseed oil (Ullah F et al. (2013) Polish J. Chem. Technol. 15: 74), chilli hazelnut oil Transesterified. The actual compositions of the reagents used are summarized in Table 9 and FIG. Detailed protocols are described in the Materials and Methods section.

[00517]

The reaction was stirred at 65 ° C. for 180 minutes to give 8.97 g of a light yellow liquid in 88% yield. The composition of the biodiesel is shown in Table 10, comparing TMSH with direct methylation using with original chili hazelnut oil.

[00519] The composition of biodiesel compared to directly methylated hazelnut oil was somewhat altered (Table 10). For methyl Z11-hexadecenoate, there was a clear reduction of 2.85%. The relative amount of other compounds, particularly long chain compounds, increased 10.63%. Overall, the process proved to be suitable for converting Z11-C16: 1 acid linked to triacylglycerides to methyl derivatives.

[00520]

2.2 Distillation of methyl (Z) -hexadeca-11-enoate
[00521] The mixture was also subjected to distillation in order to enrich the amount of methyl (Z) -hexadeca-11-enoate and to evaluate the stability of the compound under these necessary conditions. The distillation was carried out by fractionation using a 5 inch Vigreux column. The oil was heated to 210 ° C. The pressure was kept constant at 0.95 to 1.22 mbar. Over the course of 2 hours, two fractions were collected, cut I (2.21 g, head temperature 55-82 ° C.) and cut II (2.25 g, head temperature 120-146 ° C.). The composition of the different fractions, also compared to the crude mixture, is shown in Table 11.

[00522] The Vigreux column was removed prior to collecting the second fraction, as the first used device did not reach the required high head temperature.

[00523] Starting from a 23.4% initial content of methyl (Z) -hexadeca-11-enoate, it was possible to enrich the concentration to 60% by relatively simple distillation. As the 55-82 ° C temperature frame consisted of 5 different head temperatures (55-60 ° C, 63 ° C, 68 ° C, 79 ° C and 82 ° C), it is also possible to collect smaller fractions It seems that A detailed overview over the fractions is given in Table 11 and FIG.

[00524]

3. Vitride reduction of methyl (Z) -hexadeca-11-enoate
[00525] Partial reduction was then performed using a modified version of Vitride, using the fraction containing the largest amount of methyl (Z) -hexadeca-11-enoate. For this purpose, modified variants of the reagent were used. As shown in FIG. 8, the reaction comprises two steps. In the first step, the modified reducing agent has to be prepared using a nitrogen base, in this case 2,6-dimethylmorpholine. The second step uses this reagent to reduce the actual substrate.

[00526] Although not as sensitive as hydrogenated diisobutylaluminum, the reagents are sensitive to moisture, and the use of inert gases as well as dried solvents is important. Therefore, argon was used as the inert atmosphere, and only the dried solvent was used. The reaction was performed as described in the Materials and Methods section. As a crude mixture was used, the yield can not be calculated for the first reaction. A portion of the overlay of the substrate and product mixture is shown in FIG. The substrate was converted quantitatively. The only products related to methyl (Z) -hexadeca-11-enoate are 88% (Z) -hexadeca-11-enal and 12% (Z) -hexadeca-11-enol.

[00527] Products were identified by comparison of retention time with authentic standards for fragment patterns (Figures 10A-F).

[00528] Furthermore, it was observed that the 11Z-C16: 1 aldehyde, although at a very slow rate, has sufficient volatility to evaporate under full vacuum at 40 ° C. A comparison at various times during solvent removal using a rotary evaporator is shown in FIG. The same amount of substrate was used, but after an additional 2 hours under vacuum, only about 24% of the product remained. Complete removal should be avoided when carrying out the reaction. The 5.4 minute peak corresponds to hexadecanal. This observation can be used to develop relatively easy and mild purification methods.

[00529] As a result, it can be argued that Z11-hexadecenal can be produced with good selectivity by this route.

4. Plants containing a high content of "palmitobacenic acid"
[00530] In spite of the SOFA and SAG database searches already performed, the search was extended to SciFinder and Google Scholar using various minor names that appeared over time. In addition to the systematic name 11Z-hexadecenoic acid, the following minor names were also used. Lycopodium oleic acid (Arch. Pharm. 227, 241, 289, 625 (1889), which was disproved as a mixture, but later reintroduced by Riebsomer and Johnson (Riebsomer JL and Johnson JR (1933) J. Mol. Am. Chem. Soc. 55: 3352-3357)). Occasionally, as minor names, tanacetum oleic acid and palmitobactenic acid were also used, but palmitobactenic acid was the most prominent minor name.

[00531] Where possible, additional information was also collected from plant enthusiasts, plant associations, seed stores and several blogs of Wikipedia.

[00532] Grevillea exul var. rubiginosa has 45.6% of 11Z-hexadecenoic acid in the seed oil.

[00533] Kermadecia sinuata of the Proteaceae family has 40.3% of 11Z-hexadecenoic acid in the seed oil.

[00534] Orites diversifolius of the Proteaceae family has 35.6% of 11Z-hexadecenoic acid in the seed oil.

[00535] Orites revoluta of the Proteaceae family has 35.6% of 11Z-hexadecenoic acid in the seed oil.

[00536] Ziziphus jujube var. Of the Rhamnaceae family. Inermis has 33.3% of 11Z-hexadecenoic acid in seed oil.

[00537] Hicksbeachia pinnatifolia of the Proteaceae family has 28.5% 11Z-hexadecenoic acid in the seed oil.

[00538] Gevuina avellana of the Proteaceae family has 25.4% of 11Z-hexadecenoic acid in the seed oil.

[00539] Grevillea decora of the Proteaceae family has 21.4% of 11Z-hexadecenoic acid in a seed oil.

Conclusion
[00540] Almost all plants containing large amounts of (Z) -hexadeca-11-enoic acid belong to the Proteaceae family. Depending on the species and location, specimens grow from shrubs or pegs as relatively large trees.

[00541] Chilli hazel is a readily available oil source that contains large amounts (22.5%) of (Z) -hexadeca-11-enoic acid.

[00542] Transesterification / biodiesel production is an established chemical process that can be easily performed. On a 10 g scale, the reaction can be performed in 88% yield.

[00543] Small scale distillation is difficult due to the small number of theoretical plates. However, methyl (Z) -hexadeca-11-enoate can be enriched from 23.4% to 60% of the original. The data show that the configuration of the double bond is stable at high temperature for long periods of time (30 minutes at 200 ° C.), and substantial enrichment can be achieved relatively easily.

[00544] Semireduction can be performed quantitatively using 2 molar equivalents of Vitride / 2,6-dimethylmorpholine. The actual yield can not be calculated at this stage because a complex mixture was used. The results demonstrate that (Z) -hexadeca-11-enal can be produced by this route. The production distribution (relative selectivity) of compounds derived from methyl (Z) -hexadeca-11-enoate is 88% (Z) -hexadeca-11-enal and 12% (Z) -hexadeca-11- It is an enol.

[00545] In addition to plant sources, genetically engineered microorganisms (such as Candida tropicalis and Yarrowia lipolytica) that are rich in (Z) -hexadeca-11-enoate are also suitable for insect fatty alcohol / aldehyde synthesis using this method Substrate. Production of oil from microbial biomass may be more advantageous than sourcing oil from plants, as culture of the microorganism is a process that can be easily scaled up. Unlike plant cultivation, microbial culture does not depend on air and land availability, climate, irrigation, and fertilizers.

Materials and methods

[00546]

analysis
[00547] 10 μL of vegetable oil was dissolved in 450 μl of methyl tert-butyl ether in a 1.8 mL screw capped GC glass vial. Before finishing the GC-FID analysis, 50 μl of a 0.2 M solution of trimethylsulfonium hydroxide in methanol was added. This procedure is derived from Macherey-Nagel (application number 213060) based on the protocol of Butte et al., 1983 (Butte W (1983) J. Chromatogr. A 261: 142-145). The GC program used was KO-DESATUR 01. with the following parameters (Table 13): It was M.

[00548]

[00549] 10 μL of biodiesel was dissolved in 1 mL of chloroform and analyzed for biodiesel using the same GC-FID method. The Vitride reaction was analyzed by dissolving 10 μL of substrate or product mixture in 1 mL of chloroform. After the addition of 100 μL of N, O-bis (trimethylsilyl) trifluoroacetamide containing 1% trimethylsilyl chloride, the samples were analyzed by GC-FID. The MS was configured in scan mode using the same temperature program for GC-MS analysis.

Transesterification of chilli hazelnut oil
[00550] Based on the optimization protocol of Ullah et al. (2013), transesterification was performed at a methanol / TAG (triacylglyceride) molar ratio of 6: 1. In a 25 mL round bottom flask equipped with a magnetic stir bar was placed 10.22 g of chilled hazelnut oil (10.84 mmol). 1 mL of 5% NaOH in methanol was mixed with 2 mL of methanol and added to the round bottom flask. The mixture was vigorously stirred at 65 ° C. for 180 minutes. The mixture was diluted with 20 mL water before extracting twice with 20 mL methyl tert-butyl ether. The combined organics were dried over anhydrous sodium sulfate and concentrated in vacuo. The reaction yielded 8.97 g of a pale yellow liquid (yield 88%), which was analyzed by GC-FID.

Distillation of chili hazelnut biodiesel
[00551] In a 25 mL round bottom flask equipped with a magnetic stir bar was placed 8.62 g of biodiesel. The flask was fitted with a 5 inch Vigreux column, to which a water-cooled 5.5 inch short path distillation head was connected. The condenser was connected to a three-way glass receiver, to which three 10 mL glass round bottom flasks were attached. The Vigreux column was thermally insulated with aluminum foil. The head temperature was monitored using a PTFE coated T-type thermocouple coupled to a JKEM model 210 temperature controller. The pressure was monitored with a thermocouple pressure transducer (Mastercool Model 98061). A round bottom flask containing seed oil methyl ester was placed in an oil bath and carefully heated to 210 ° C. while stirring with a magnetic hot plate stirrer. Two different fractions were collected over the course of 2 hours. The first fraction (cut I-2.21 g) was collected at a temperature range of 55-80 ° C., and the second fraction (cut II-2.25 g) was collected at 120-146 ° C. The Vigreux column could not be used to reach the high head temperature of Cut II, so it was removed before collecting the second fraction. After the distillation procedure, 3.54 g remained in a 25 mL round bottom flask. The different fractions were analyzed by GC-FID.

Vitride reduction of the first cut-crude fraction containing the highest content of methyl (Z) -hexadeca-11-enoate
Preparation of reducing agent (cis-2,6-dimethylmorpholine modified red-Al)
[00553] A dry 3-neck flask was equipped with a dry addition funnel, a magnetic stir bar and a septum. After four pump purges with argon, the flask was placed in an ice bath. 14 mL of anhydrous tetrahydrofuran and 1.17 mL (9.54 mmol) were charged. After the mixture is allowed to cool to 0 ° C., a 3.5 M solution of sodium bis (2-methoxyethoxy) aluminum hydride (2.48 mL, 8.67 mmol) in toluene is added dropwise and stirred for 1 hour to give a colorless, clear, homogeneous solution. I got

[00554] Partial reduction of the cut I of chili hazelnut by diesel
[00555] A dry 3-neck flask was fitted with a dropping funnel, a magnetic stir bar and a septum. After four pump purges with argon, the flask was placed in an ice bath. 20 mL of anhydrous tetrahydrofuran and 0.97 g of chilli hazelnut by diesel cut I (3.81 mmol, estimated to be 11Z-C16: 1-COOMe only) were added. The previously prepared cis-2,6-dimethylmorpholine modified Red-Al was attached to the dropping funnel by cannula transport. After the mixture was cooled to 0 ° C., modified Red-Al was added dropwise (reagents were not cooled at this stage). The mixture was stirred for 30 minutes and allowed to return to room temperature. The reaction was quenched with 80 mL of 2N hydrochloric acid and the product was extracted twice with 50 mL of methyl tert-butyl ether. The combined organics were dried over anhydrous sodium sulfate and concentrated in vacuo. The reduced mixture was analyzed by GC-MS, and the product derived from methyl (Z) -hexadeca-11-enoate was identified by comparison with the authentic standard for retention time and fragment pattern.

Example 3: Expression background and rationale for transmembrane desaturase in Yarrowia lipolytica
[00556] Engineering the microbial production of unsaturated lipid moieties requires functional expression of synthetic pathways. One such pathway involves transmembrane desaturases that mediate the conversion of fatty acyl CoA to regiospecific and stereospecific unsaturated fatty acyl CoA. Many genes encoding these enzymes have been found in some insects as well as some microalgae. Regiospecific and stereospecific desaturases can be used to produce microbial oils rich in fatty acid precursors. The microbial oil can then be derivatized and reduced to the active ingredient. A number of genetic variants were screened to identify enzyme activities that would allow the creation of pathways capable of high level synthesis of singles or blends of unsaturated lipid moieties. Furthermore, to optimize the search towards finding suitable hosts for optimal expression of these transmembrane proteins, these enzymes across a large number of hosts (Saccharomyces cerevisiae, Candida viswanathii (tropicalis), and Yarrowia lipolytica) Was screened.

[00557] S. S. cerevisiae and C.I. By initial screening of the desaturases in Viswanathii (tropicalis), Amyelois transitella, Helicoverpa zea, and Trichoplusia ni. From which three active Z11-C16: 1 desaturase variants were identified. S. For screening of S. cerevisiae, S. coli fused to the full-length insect Z11 desaturase sequence. The coding sequence with the N-terminal leader sequence of S. cerevisiae Ole1p Z9 desaturase was used. This strategy was previously described by S. It has been used in the scientific literature to express eukaryotic desaturases in S. cerevisiae. All three of the above desaturases showed Z11 desaturase activity with an Ole1p leader fusion when expressed with an OLE1 deletion background. C. A similar design, including the albicans Ole1p leader sequence, was prepared according to Used for zea's Z11 desaturase. Although active, this Ole1 p-H. The zea desaturase fusion did not significantly increase the titer of Z11-hexadecenoic acid. Furthermore, conservatively optimized A. The transitella Z11 desaturase is S. cerevisiae and C.I. It was active in both viswanathii. The following studies show two different Y.. In H. lipolytica strains, namely SPV140 and SPV300. zea, T. ni, and A. We focused on testing the functional expression of Z. H. zea and T. For the ni desaturase, using native and Homo sapiens codon optimization sequences Only the native sequence was used for transitella. Finally, Y. The N-terminus of lipolytica Ole1p Z9 stearoyl CoA desaturase is S. S. cerevisiae or C.I. The insect desaturases are more closely aligned than the N-terminus of the albicans Ole1 p. Based on this alignment, two additional desaturase versions were generated. The putative leader sequence is Y.3. lipolytica Ole1p to T. l. ni and H. It was replaced with zea desaturase.

Summary of approach
[00558] S. S. cerevisiae or C.I. A focused library of Z11 desaturases (Insect origin: Amyelois transitella, Helicovera zea, Trichoplusia ni) whose activity was observed in any of viswanathii, cloned into a dual crossover cassette targeting the XPR2 locus with URA3 selectable markers did. The protein coding sequence may be the natural insect sequence (SEQ ID NO: 17, 18), Homo sapiens optimized coding sequence (SEQ ID NO: 19, 20), or the N-terminal 84 bases (H. zea, SEQ ID NO: 22) or 81 bases (T ni, SEQ ID NO: 21). Use any of the Homo sapiens optimized sequences exchanged with the N-terminal 96 bases of the lipolytica OLE 1 (YALI0C05951) gene. S. S. cerevisiae and C.I. Unlike in the case of viswanathii screening, in leader sequence chimeras, instead of linking the host leader sequence to the N-terminus of the full length desaturase coding sequence, direct exchange of leader sequence is considered. A. Only the native coding sequence was used for the transitella desaturase (SEQ ID NO: 23).

[00559] Each of the seven desaturase constructs was transformed into SPV140 (PO1 f) and SPV300 (H222 ΔP ΔA ΔF ΔURA3) to confirm site-specific integrants.

[00560] Desaturase activity was tested by in vivo bioconversion of hexadecanoic acid (palmitic acid) to (Z) -11-hexadecenoic acid (palmitobacenic acid) in YPD medium.

[00561] GC-FID analysis was used for identification and quantification of metabolites.

Construction of the resulting strain
[00562] Desaturase variants are targeted to insertion into the XPR2 locus. It was cloned into the pPV101 vector containing the lipolytica expression cassette.

[00563] T. ni and H. The desaturases of zea, respectively, can be either the natural insect sequence (SEQ ID NO: 17, 18), the full-length insect sequence codon-optimized for Homo sapiens (SEQ ID NO: 19, 20), or It synthesize | combined using the putative leader sequence (sequence number 21, 22) substituted by the leader sequence of lipolytica OLE1 desaturase. A. The transitella desaturase was also synthesized using the natural insect coding sequence (SEQ ID NO: 23). All seven desaturase variants were transformed into SPV140. Based on the previous activation results, H. zea and A. a. Only transitella desaturase variants were transformed into SPV300.

Functional expression assay
[00564] Functional activity, C.. It was evaluated by modifying the protocol used for transmembrane desaturase expression in Viswanathii SPV 053. Briefly, Y. pylori expressing insect desaturase. Strains derived from L. lipolytica SPV140 and SPV300 were cultured in rich (YPD) to produce biomass. Using biomass produced by YPD, in a small scale (2 ml) culture containing palmitic acid, cultured in a deep 24 well plate for a total of 48 hours (see Materials and Methods for details).

[00565] T. ni, H. zea, and A. In the initial screening of variants of transitella, H. coli codon-optimized for Homo sapiens. Only the zea desaturase variants produced detectable Z11-hexadecenoic acid (FIG. 12). Natural H. Expression of zea desaturase results in the production of 100 ± 5 mg / L of Z11-hexadecenoic acid, as described by Y. et al. The version with the lipolytica OLE 1 leader sequence produced 83 ± 11 mg / L. As seen in FIG. 12, the distribution of the other major fatty acid species was relatively unaffected by functional desaturase expression. In the active strain, Z11-hexadecenoic acid accounted for about 10% (g / g) of the fatty acid species (including palmitic acid substrate that can be adsorbed to the outer cell surface).

[00566] Follow-up experiments were performed comparing activity variants in the SPV 140 background with SPV 300 derived desaturase strains. Parental SPV300 and SPV140 expressing hrGFP were used as negative controls. The same bioconversion assay protocol was used. As with SPV 140, H.1. Only the sapiens optimization variant produced detectable activity (FIG. 13). The SPV300 strain grew to a higher final cell density (SPV300 OD600 = 26-28), SPV140 OD600 = 19-22) (FIG. 14). The maximum titer is H.1. H. sapiens codon optimized natural H. coli. It was observed in a strain expressing zea Z11-desaturase (pPV199). The retested SPV 140 strain produced 113 ± 1 mg / L (5.5 ± 0.2 mg / L / OD) of Z11-hexadecenoic acid, which was 13% greater than the titer observed in the first experiment (FIG. 15) . The SPV 300 strain, which expresses the same desaturase, produced a wider range of productivity. They produced on average 89 ± 18 mg / L (3.3 ± 1.2 mg / L / OD) of Z-11 hexadecenoic acid, but one clone is 124 mg / L (4.6 mg / L / OD) of Z11-hexadecene The acid was produced (Figure 15).

[00567] In summary, H. Only zea Z11 desaturase variants produced detectable Z11-hexadecenoic acid. Under the current assay conditions, a slightly higher titer was observed against SPV300 at SPV140 background. The Z11-hexadecenoic acid titers are summarized in Table 14.

[00568]

[00569] In SPV 300, one non-site specific integrant (Y. lipolytica OLE1-H. Zea Z11 desaturase with Homo sapiens codon optimization) was tested for pPV200. The integrant did not produce detectable Z11-hexadecenoic acid, but the two site-specific integrants produced 55 ± 1 mg / L.

[00570] No major hydroxy or diacid peak is observed from pellets of SPV140 or SPV300 derivatives, and the absence of beta oxidation / omega oxidation genes in SPV300 is the current assay conditions (relatively low substrate concentrations, rich media) Below did not increase the accumulation of Z11-hexadecenoic acid.

Conclusion
[00571] H. zea Z11 desaturase is active, resulting in the production of about 100 mg / L of Z11-hexadecenoic acid from about 500 mg / L of palmitic acid substrate. Functional expression was demonstrated from repeated experiments in three positive integrant and 24-well plate assays.

[00572] H. The zea desaturase is Y. Codon optimization (Homo sapiens or possibly Y. lipolytica) was required for activity in lipolytica.

[00573] T. The ni Z11 desaturase is S. active in S. cerevisiae but Y. It does not produce detectable Z11-hexadecenoic acid in lipolytica.

[00574] Y. The reproducibility of the assay for L. lipolytica strains can be confirmed starting from glycerol stocks.

[00575] A. The transitella desaturase is Y.. It can be codon optimized for expression in lipolytica.

[00576] Y. As L. lipolytica is a candidate production host, additional copies of the active desaturase were isolated from Y. coli. It can be inserted into lipolytica, culture conditions to improve bioconversion can be established, and substrate conversion can be quantified.

Construction of materials and method stocks
[00577] All desaturase genes were synthesized (Genscript). Either native sequence or Homo sapiens codon optimization was used. The synthesized gene was subcloned into pPV101. The plasmids were transformed into E. coli using the Zyppy Plasmid Miniprep kit (Zymo Research, Irvine, CA). E. coli EPI 400 was transformed and prepared. Approximately 1-2 μg of linearized DNA was transformed using the Frozen-EZ Yeat Transformation II kit (Zymo Research, Irvine, Calif.). The entire transformation mixture was placed on CM-glucose-ura agar plates. Positive integrants were found to be site specific and were genotyped by check PCR.

Functional expression assay
Palmitic acid supplementation in YPD
[00579] Positive isolates were repatched on YPD, grown overnight and then stored at 4 ° C. Strains were inoculated from patch plates into 2 ml YPD in deep 24 well plates (square wells, pyramidal bottom). Three positive clones were inoculated for each desaturase variant. Three isolates of pPV101 in SPV140 and parent SPV300 were used as negative controls. The deep well plates were incubated at 28 ° C., 250 rpm for 24 hours in an Infors Multitron refrigerated flask shaker. After 24 hours of incubation, a volume of 1 ml of each culture was pelleted by centrifugation at 500 × g. Each pellet was resuspended in 2 ml of YPD. From a 90 g / L stock solution in ethanol, 500 mg / L palmitic acid was added to the culture. As a result, 0.5% ethanol containing palmitic acid substrate was added. All cultures were incubated for 48 hours before endpoint sampling. The final cell density was measured using a Tecan Infinite 200pro plate reader. 0.75 ml or 0.8 ml of each culture was transferred to a 1.7 ml microfuge tube and pelleted. The supernatant was removed and the pellet was processed as follows.

Metabolite extraction and GC-FID analysis
[00580] The total lipid composition and the determination of (Z) -11-hexadecenoic acid are described by Moss et al. (1982) (Moss, CW, Shinoda, T, Samuels, JW Determination of cellular fatty acid compositions of various yeasts by gas-liquid chromatography. J. Clin. Microbiol. 16: 1073-1079 (1982) and Yousuf et al. (2010) (Yousuf, A., Sannino, F., Addorisio, V., Pirozzi, D., Microbial Conversion of Olive Oil Mill Wastewaters into Lipids Suitable for Biodiesel Production. J. Agric. Food Chem. 58: 8630-8635 (2010)). Typically about 10 mg to 80 mg of pelleted cells (in 1.5 mL plastic tubes) were resuspended in methanol containing 5% (w / w) sodium hydroxide. The alkaline cell suspension was transferred to a 1.8 mL crimp vial. The mixture was heated at 90 ° C. for 1 hour in a heat block. The vial was allowed to cool to room temperature prior to acidification with 400 of 2.5 N HCl. 500 μL of chloroform containing 1 mM methyl heptadecanoate was added, the mixture was vigorously stirred and then both the aqueous and organic phases were transferred to a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm and then 450 μL of the organic phase was transferred to a GC vial. For lipid analysis and fatty acid determination, 50 μL of 0.2 M TMSH (trimethylsulfonium hydroxide in methanol) was added and samples were analyzed by GC-FID.

Example 4: Production background and rationale for Z11-14 acid in Yarrowia lipolytica
[00581] Yarrowia lipolytica was engineered to produce Z11-14 acid, which is a precursor of the target Lepidoptera pheromone Z11-14 Ac.

[00582] A library of 73 desaturases was selected to target possible pheromone including Z11-14 Ac, Z7-12 Ac, Z9 E12-14 Ac, E8 E10-C12 OH and Z9 E11-14 Ac. All desaturases were tested in the H222 ΔP ΔA ΔF (SPV 300) background.

[00583] Eleven desaturases have been determined from the literature to have Δ11 activity (DST 001 to DST 009, DST 030, and DST 039, Table 15). All desaturases were screened by feeding methyl palmitate (C16), methyl myristate (C14), or methyl laurate (C12) as a substrate, and the overall product profile was determined by GC analysis.

[00584] The activities obtained from the Δ11 desaturase library, and from other desaturases that have been shown to produce Δ11 compounds, in particular Z11-14 acid, are discussed.

[00585]

result
[00586] When 2 g / L of methyl myristate was supplied to the desaturase library, production of up to 69 mg / L of Z11-14 acid was observed (Figure 16). The currently best desaturase Helicoverpa zea (Hz) DST (amino acid sequence of SEQ ID NO: 1, SPV 459 encoded by the nucleotide sequence of SEQ ID NO: 20) is 16 mg / L in addition to desaturases DST 001 to DST 007, DST 030 and DST 039 An amount of Z11-14 acid in the range of 69 mg / L is produced. DST 001 (A. velutinana), DST 004 (M. sexta), and DST 039 (A. transitella) are more specific for the production of Z11-14 acid than for Z11-16 acid, but these desaturases are approximately 20 mg / L Z11-14 acid is produced. Strains producing higher Z11-14 acid titers also produced Z9-14 acid from methyl myristate substrate at 20-30 mg / L, but this is less compared to negative control SPV298. The C14-C18 production profile of Hz DST (SPV 459) compared to SPV 298 is shown in FIG.

[00587] Figure 12B shows the proof of concept for the synthesis of Z11-14 acid. An attempt was made to identify enzymes with improved Z11-16 acid titer or production specificity compared to Helicoverpa zea DST (Z11-16 acid 1.05 g / L; Z11-14 acid 69 mg / L). There was no desaturase with higher production than Hz DST (SPV 459), but DST 003 (SEQ ID NO: 2) has a production phenotype similar to the HzDesat strain, and DST 002 and DST 005 reduced with similar Z11-16 acid production It had Z11-14 acid production. The desaturase of DST 006 was expressed in SPV 459. Genetically equivalent to zea desaturase and served as a library control. DST 006 produced equivalent levels of Z11-16 acid by feeding methyl palmitate, but this strain produced lower titers of Z11-14 acid with methyl myristate. Genetic changes in the strain background may be the cause of the observed differences.

[00588] DST039 (A. transitella) was previously screened under different conditions. In the rich culture medium, natural A. The production of Z11-16 acid by the transitella coding sequence was not observed. H. Although sapiens optimized sequences were tested, no activity was also observed under rich media conditions. In this screening, DST 039 was tested by Hs optimized sequencing under nitrogen limited conditions and resulted in the production of 235 mg / L Z11-16 acid and the production of 21 mg / L Z11-14 acid on the corresponding substrate.

[00589] The Δ11 product from DST 008 (Drosophila melanogaster) or DST 009 (Spodoptera littoralis) in the SPV 300 background was not observed.

Overview
[00590] The production of Z11-14 acid was observed with 10 desaturases at titers ranging from 16 mg / L to 69 mg / L (supplied with methyl myristate 2 g / L).

[00591] (The amino acid sequence of SEQ ID NO: 1, encoded by the nucleotide sequence of SEQ ID NO: 20) zea DST was still the best Z11-16 acid-producing strain (> 1 g / L with methyl palmitate supply).

[00592] DST 003 (S. inferens, SEQ ID NO: 2) has a H. It has the most similar phenotype to zea DST.

[00593] DST 002 (S. litura) and DST 005 (O. nubialis) have been described for H. coli as to production of Z11-16 acid. It is more specific than zea DST (reduced production of Z11-14 acid).

[00594] DST 001 (A. velutinana), DST 004 (M. sexta), and DST 039 (A. transitella) have been described for H. coli as to production of Z11-14 acid. It is more specific than zea DST.

Conclusion
[00595] Z11-14 acid can be produced by heterologous expression of a specific desaturase in Yarrowia lipolytica if methyl myristate is provided.

[00596] Multiple copies of desaturase (identical sequence or combination of sequences) are inserted into the improved strain background for increased Z11-14 acid titer, product specificity, and genetic stability.

Material and Method Library Generation
[00597] Desaturase sequences were provided to Genscript for codon optimization (Homo sapiens expression organisms) cloning into pPV266 (XPR2 locus insertion vector with TEF promoter and terminator) using Pacl / Sapl restriction digest. Lyophilized DNA as well as EPI 400 agar stubs were provided. The desaturase constructs are summarized in Table 16.

[00598] The construct was linearized using Pmel restriction enzyme and directly transformed into host strain SPV300. Transformants were verified by check PCR using primers internal to the pTEF promoter outside the XPR2 insertion junction.

[00599]

Plasmid digestion
[00600] Approximately 10 μg of lyophilized DNA was ordered from Genscript. The DNA was resuspended in 50 μL water to a final concentration of approximately 200 ng / μL. 10 μL of DNA was mixed with 1.25 μL of 10 × CutSmart Buffer and 1.25 μL of Pmel restriction enzyme (reaction volume 12.5 μL). The reaction was incubated for 1.5 hours at 37 ° C. in a PCR device and heat inactivated at 65 ° C. for 30 minutes.

Transformation
[00601] SPV300 competent cells were grown by inoculating YPD cultures at 0.001 OD in a baffled flask and grown to 0.5-1.0 OD. The cells were harvested at 800 × g and washed with 0.25 volumes of solution 1 of the Zymo Frozen-EZ Transformation II kit for yeast. The cells were resuspended in solution 2 at a 1000-fold original culture volume and frozen slowly at -80 ° C while adiabatic in a Styrofoam container (freeze cells may have better transformation efficiency than fresh cells) . First, 50 μL of cells were mixed with 12.5 μL of the digestion reaction (no cleanup required) and then with 500 μL of solution 3. The transformants were incubated at 28 ° C. for 3 hours without shaking, after which the complete transformation mixture was plated on appropriate selective agar medium. The petri dishes were incubated for 3 to 4 days until colonies appeared.

Check PCR
[00602] Transformed colonies were picked and transferred to 7 μL water in PCR plates. 5 μL of cells were patched with multiple channels, transferred to selective omnitrays and allowed to grow overnight. The remaining 2 μL of cells were microwaved for 2 minutes and 15 μL of PCR master mix was added.

[00603] PCR cycle

Colony patching
[00604] Positive clones were repatched into a 24-well format YPD omnitray containing assay controls. Omnitrays were grown overnight at 28 ° C. and used to inoculate a bioassay culture.

Bioassay
[00605] Positive transformants (N = 4 clones per construct) were inoculated into 1 mL of YPD in a 24-well culture plate and incubated for 24 hours with Infors HT Mulitron Pro while shaking at 1000 rpm at 28 ° C. The cells were pelleted at 800 × g and resuspended in S2 medium containing 5 μL of substrate (concentration about 2 g / L). After 48 hours of bioconversion, 250 μL of culture was harvested into glass crimp top vials.

S2 medium
[00606] 2 g / L yeast extract, 1 g / L peptone, 0.1 M phosphate buffer, aa, no NH4 1.7 g / L YNB, 60 g / L glucose, 5 g / L glycerol

GC sample processing
[00607] Front entrance / detector system 6890 GC, ChemStation Rev. B. 03.02 (341)
Column J & W DB-23 30m x 25mm x 25μm
Runtime = 14.4 minutes inlet Heater = 240 ° C, pressure = 9.0 psi, total flow {H2} = 36.2 mL / min Carrier H2 @ 1.0 mL / min, 9.0 psi, 35 cm / sec Signal Data rate = 2 Hz /0.1 minute oven 150 ° C, 1 minute
Gradient 12 ° C / min to 220 ° C, 3 minutes holding
Gradient 35 ° C / min to 240 ° C, holding 4 minutes
Equilibration time: 2 minutes injection split, 240 ° C
Split ratio-30: 1; 29.1 mL / min Detector FID, 240 ° C
H2 @ 35.0 mL / min, air @ 350 mL / min
Electrometer {Lit offset} @ 2.0 pA
Sample injection volume = 1 μL

[00608] Back inlet / detector system 6890 GC, ChemStation Rev. B. 03.02 (341)
Column J & W DB-23 30m x 25mm x 25μm
Runtime = 14.4 minutes inlet Heater = 240 ° C, pressure = 9.8 psi, total flow {H2} = 40.1 mL / min Carrier H2 @ 1.1 mL / min, 9.8 psi, 38 cm / sec Signal Data rate = 2 Hz /0.1 minute oven 150 ° C, 1 minute
Gradient 12 ° C / min to 220 ° C, 3 minutes holding
Gradient 35 ° C / min to 240 ° C, holding 4 minutes
Equilibration time: 2 minutes injection split, 240 ° C
Split ratio-30: 1; 32.3 mL / min Detector FID, 240 ° C
H2 @ 35.0 mL / min, air @ 350 mL / min
Electrometer {Lit offset} @ 2.0 pA
Sample injection volume = 1 μL

[00609] TMSH: Trimethylsulfonium hydroxide (0.2 mol / L in methanol)-VWR TCT 1576-025 ML

Example 5: Background and rationale for converting fatty acid methyl ester (Z11-16 FAME) precursor to Z11-16 Ald
[00610] Z11-16Ald is one of the target active ingredients (AI) in the present disclosure. Research and development on the synthesis of this AI has four major areas: Biocatalyst engineering operation, 2. Fermentation process development, 3. Downstream process (DSP) development (conversion of the microbially produced Z11-16 precursor to purified Z11-16FAME), 4. Central to the final chemical conversion of the FAME precursor to the target AI (acetate or aldehyde).

[00611] DSP comprises converting a microbial lipid comprising Z11-16 acid to Z11-16 FAME, and subsequent enrichment by distillation and chemical conversion to Z11-16 Ald. A description of the designed process to obtain crude Z11-16 FAME is shown in FIG. First, we searched for multiple choices of DSP at each stage. The most efficient methods and compatibility with industrial equipment at each stage were determined. The choice of method was also evaluated based on the estimated cost contribution in the overall manufacturing cost of pheromone.

[00612] This example focuses on supporting the concept of final chemical conversion of Z11-16 FAME precursors to Z11-16 Ald.

Generation of result Z11-16 FAME
[00613] 1. Heat-killed fermentation, and Cell separation
[00614] The fermentation culture of SPV 459 was heated to 70 ° C and held for 3 hours to heat kill the cells and inactive lipase. A filter aid was added prior to filtration. A filter cloth was attached to the Seitz filter casing. Immediately after filling the Seitz filter and applying a pressure of 0.5 bar, about 250 mL of the first filtrate was collected and filtration was stopped. Cloudy filtrate was again met in Seitz filter, was continued filtered through a _{N 2} of 1bar.

[00615] 3. Extrusion
[00616] The cake was pressed against the feathers and the developing yeast pellet was scraped off with a spatula to form a pellet.

[00617] 4. Drying
[00618] Drying was done in a ventilated oven. The pellets were uniformly distributed in monolayers on metal trays and dried in a ventilated oven at 36 ° C. for 20 hours.

[00619] 5. TAG extraction, 6. Debris separation, 7. evaporation
[00620] A 500 mL three-necked flask was charged with 65 g of cell pellet and fitted with a mechanical stirrer, thermometer, and reflux condenser. After addition of 330 mL n-heptane, the mixture was heated to 70 ° C. and stirred at 40 rpm. The mixture was kept at 70 ° C. for 3 hours. The still hot mixture was filtered directly through a Seitz pressure filter using the same 20 micron filter cloth used for the initial cell filtration. After the first filtration, the empty three-necked flask was flushed with 50 mL n-heptane. The filter was flushed with wash heptane and collected in the same flask as the first filtrate. After filtration, the pellet was returned to the same flask as before and the filtrate was evaporated at 50 ° C. under reduced pressure. The extract was weighed and the extract sampled for GC and acid number analysis.

[00621] For the second extraction step, 250 mL of n-heptane was added and the mixture was stirred for 1.5 hours. Workup and sampling were the same as in the first extraction step. The third extraction was performed almost the same as the second extraction except that the extraction time was extended to 2 hours.

[00622] 8. Esterification, 9. Transesterification reaction, 10. Separation / evaporation, 11. Drying
[00623] A 500 mL three necked flask was charged with 17.36 g of TAG extract and 100 ml of methanol and fitted with a mechanical stirrer, thermometer, and reflux condenser. After adding 0.506 g of p-toluenesulfonic acid monohydrate (2.66 mmol), the mixture was heated to 60 ° C. (setting temperature 60 ° C., actual temperature 53.8 ° C.) and stirred at 180 rpm. After reaching the final temperature, the stirring was increased to 240 rpm and 3 mL of trimethyl orthoacetate (23.57 mmol) was added. The reaction was monitored by TLC. After 60 minutes, TLC showed that all the palmitic acid residue had been converted. An additional 2 mL of trimethyl orthoacetate (15.71 mmol) was added before 1 mL of sodium methoxide solution (25% in methanol, 4.36 mmol) was added. The mixture was stirred for a further 80 minutes and monitored by TLC. After all the TAG had been converted, the reaction was quenched with 0.4 mL of acetic acid. The mixture is extracted with 100 ml of n-heptane. The organic phase is separated and the aqueous phase is extracted with a further 20 mL of n-heptane. The combined organic phases were washed with 30 mL dH2O. The organic phase is concentrated in vacuo.

[00624] 12. distillation
[00625] A 100 mL round bottom flask was filled with 30 g of crude FAME. A magnetic stir bar was placed in the contents of the pot. To fractionate the distillate, the flask was fitted with a Vigreux column and a microdistillation variable reflux distillation head was connected. The distillation head was fitted with a thermometer, a condenser, a collection flask, and a vacuum line. The device was thermally insulated. The distillation was performed at a temperature of up to 250 ° C. under a reduced pressure of about 500 mtorr. The various fractions were collected and analyzed by GC.

reduction
[00626] Briefly (Bazant, V. et al., "Properties of sodium-bis- (2-methoxyethoxy) aluminumhydride. I. Reduction of some organic functional groups.", Tetrahedron Letters 9.29 (1968): 3303-3306) and A dropping funnel, magnetic stir bar, and septum were attached to a dry three-necked flask according to the protocol of (Unauthored (2014)). After the flask was purged with argon, the flask was placed in an ice bath. The flask was charged with 10 mL of anhydrous tetrahydrofuran and 2.36 g of Z11-16FAME (approximately 70% purity). The addition funnel was charged with 20 mL of anhydrous tetrahydrofuran and 3.13 mL (21.98 mmol) of 60% SMEAH reagent in toluene. After cooling the mixture to 0 ° C., the SMEAH solution was added dropwise over 30 minutes. After addition of SMEAH, the ice bath was replaced and the mixture was stirred for 4 hours. The reaction was quenched with 5 mL of 10% sulfuric acid and the product was extracted twice with 20 mL of methyl tert-butyl ether. The combined organics were dried over anhydrous sodium sulfate and concentrated in vacuo.

TEMPO bleach oxidation of Z11-hexadecenol to Z11-hexadecenal
[00627] 50 mL of buffer solution (NaHCO of 10.08 g _3, 15.3 g of Na ₂ HPO _4, and 1.44g dissolved NaH ₂ PO ₄ in H ₂ O in 500mL of, pH = 7.8) in a glass beaker 250mL including , 30 mg of toluene containing tetrabutylammonium hydrogen sulfate (TBAH) (407 mg), 2.89 g of Z11-hexadecenol (purity 54.3%), and 1 mL of HO-TEMPO solution (207 mg / mL in toluene) were added. The mixture was stirred at 4000 rpm for 30 seconds with a Silverson mixer, then 25 mL of 4.4% bleach solution (pH 8.58) was added via syringe pump in 90 seconds. While stirring, the temperature and pH of the reaction were monitored over 8 minutes. After stirring for 8 minutes, 550 μL of concentrated HCl was added to neutralize the reaction solution, and the measurement pH was adjusted to 7.06. The reaction mixture was transferred to a separatory funnel and extracted with toluene. The organic layer was collected and concentrated in vacuo.

[00628] The GC-FID trace of the Z11-hexadecenol starting material is shown in FIG.

[00629] The pH and temperature over the course of the oxidation reaction are shown below.

[00630] The GC-FID trace of the oxidation product is shown in FIG.

[00631]
The reaction results are shown below.

[00632] Calculation of product concentration

[00633]

[00634] Calibration curve based on 1-tetradecanol internal standard

[00635] Analysis
[00636] All samples were analyzed on an Agilent 6890 GC equipped with a J & W DB-23 column. The following temperature program was used for analysis of bleached oxidized samples. Hold at 100 ° C. for 1 minute, ramp 5 ° C./min to 200 ° C., hold 3 min, ramp 35 ° C./min to 240 ° C., hold 10 min. All other samples were derivatized with KOH / H ₂ SO ₄ and analyzed with the following program. Hold at 150 ° C. for 1 minute, ramp 12 ° C./min to 220 ° C., hold 3 min, ramp 35 ° C./min to 240 ° C., hold 4 min.

Example 6: Improved Z11-16 acid producing strain background and rationale
[00637] Screening of a small library of Z11-specific insect desaturases allows variants of Helicoverpa zea to be isolated from Y. coli on 16 acid substrates. It was identified as having the highest activity on L. lipolytica H222 ΔP ΔA ΔF background (see Example 3). H. Three integrants of zea desaturase were screened and stored in the strain database as SPV458, SPV459, and SPV460. In the first screen, SPV 459 produced higher titers than sister strains SPV 458 and SPV 460 and was selected for further process development. SPV 459 was also initially selected for further strain engineering, but the selectable marker could not be stably removed from the strain. SPV 458 was chosen to replace SPV 459 for the purpose of engineering engineering of the strain, and the marker was successfully rescued from SPV 458. A total of 22 plasmid constructs were inserted into SPV 739 and markers rescued the SPV 458 descendents. H. Strains with additional copies of zea desaturase produced increased Z11-16 acid titers. Four of these strains were evaluated for improved performance in the Biolector small scale assay and DASGIP 1L fermentor. Strains with additional desaturase copies performed better than their SPV 458 parent but did not exceed SPV 459. Based on these results, two strains, SPV 968 and SPV 969, were selected for further engineering operations. These two strains were marker rescued and an additional DNA cassette was inserted to create a small library of strains with two or three copies of the Z11 desaturase and lacking key lipid metabolism genes ( Figure 21).

[00638] Several further changes were made to improve the Biolector assay. While the performance of the strain is generally predictable on the 1 L scale, lower cell densities and maximal titers were observed in the Biolector assay. The semi-defined media used in the fermentation batch process v1.1 (SOP 026.2) was selected to increase cell density, improve titer and improve correlation with fermentation performance. Current fermentation processes also use 32 ° C. incubation temperatures. Temperatures of 28 ° C. and 32 ° C. were compared using fresh medium. Finally, current fermented feed batch processes employ an initial batch phase of high glycerol concentration and low glucose concentration. An alternative Biolector batch medium was tested using 60 g / L glycerol and 40 g / L glucose.

[00639] An improved Z11-16 acid B _d (biodesaturation) strain was identified by gene deletion of the lipid mobilization gene and insertion of additional copies of Z11-16 acid desaturase.

[00640] examined the response of improved Z11-16 acid B _d strains to differences cosubstrate composition (concentration of glycerol and glucose).

approach
[00641] Either Helicoverpa zea desaturase (amino acid sequence of SEQ ID NO: 1, codon optimized nucleotide sequence of SEQ ID NO: 20) or Sesamia inferens Z11 desaturase (amino acid sequence of SEQ ID NO: 2, codon optimized nucleotide sequence of SEQ ID NO: 39) A linear DNA cassette that expresses A lipolytica H222 ΔP Δ A ΔF (SPV 298) derivative was inserted at multiple loci.

[00642] The resulting strains were assayed at 700 μl bioconversion using a 48-well flower plate in M2P Labs Biolector.

[00643] Two incubation temperatures, 28 ° C and 32 ° C, were tested.

[00644] Two media compositions were tested for subsets of strains. The media were identical except for the content of glucose and glycerol. Both media were 1.7 g / L yeast nitrogen base without ammonium sulfate or amino acids, 1 g / L yeast extract, 3.3 g / L ammonium sulfate, 100 mM potassium phosphate buffer, and 8.5 mg / L Contained iron sulfate. A total of 20 g / L 16ME (methyl palmitate) was added as two 10 g / L boluses. The co-substrate concentrations were as follows.
[00645] High glucose primary medium: 120 g / L glucose and 5 g / L glycerol
[00646] High glycerol medium: 40 g / L glucose and 60 g / L glycerol
[00647] The growth rate, specific productivity of Z11-16 acid, maximum Z11-16 acid titer, and Z11-16 acid selectivity were compared between strains.

[00648] Selectivity is defined as follows.

[00649] Here, g16 acid _S is an unconverted substrate, and g16 acid _I is a 16 acid which is internalized and stored as a triacylglyceride or another intracellular lipid.

result
[00650] In addition to the first generation controls SPV 458 and SPV 459, nine second and third generation strains were tested. All constructs are as described in H. One copy of zea Z11 desaturase and S. coli identified in the first generation desaturase library (DSTg1). With the exception of SPV 1060 containing one copy of the inferens Z11 desaturase, It contained a copy of zea Z11 desaturase (see Example 4). Several strains also contained the loss of key genes in lipid metabolism, FAT1 (fatty acyl-CoA synthetase / fatty acid transporter), TGL3 (intracellular triacylglycerol lipase), and GUT2 (glycerol-3-phosphate dehydrogenase) . Gut2p dehydrogenase catalyzes a second reaction in the glycerol utilization pathway, converting glycerol-3-phosphate to DHAP. GUT2 knockouts grown in the presence of high concentrations of glycerol can result in the accumulation of high concentrations of glycerol-3-phosphate which may dysregulate catabolism. This was observed in the screening of second generation strains. In order to avoid this growth inhibition, GUT2 deficient strains were grown in medium without glycerol (see Materials and Methods).

[00651] Comparison of culture temperature at 28 ° C and 32 ° C in high glucose medium (HSD036, HSD043, HSD055, HSD056, and HSD061)
Growth rate
[00653] Five independent experiments were performed to test bioconversion in high glucose medium at both 28 ° C and 32 ° C. All 11 strains were tested under at least one temperature condition. Initial growth rates were similar regardless of genotype (FIG. 22A). The growth rate at 28 ° C. was 0.21 to 0.23 h ⁻¹ . At 32 ° C., the growth rate was somewhat faster for some strains, with an overall range of 0.23 to 0.24 h ⁻¹ . Strains incubated in high glucose medium without glycerol used in HSD 043 grow at a slower rate (0.13 to 0.14 h ^-1 ), and even 5 g / L of glycerol can significantly affect the growth rate (Figure 23).

[00654] Specific productivity
[00655] In high glucose media, maximal specific productivity (gZ11-16 acid / L / OD-h) was observed at the first sampling at 23 hours (substrate addition at 7 hours). In some cases, particularly at 32 ° C., the specific productivity was also maintained at later times. At 28 ° C., both second and third generation strains derived from SPV 458 showed higher specific productivity than SPV 459 (FIG. 22B). For the first two desaturase copies, a nearly linear dependence of the specific productivity on the desaturase copy number (about 2 mg / L-OD-h-copy number) was observed. The addition of the third desaturase copy increases the specific productivity slightly.

[00656] At 32 ° C, higher specific productivity was observed for all strains except for 3 strains SPV1056, SPV1 199, and SPV1200 (Figure 22B, Figure 24B). All three of these strains are H.. It is a strain with 3 copies of zea desaturase, all containing FAT1 deletion. SPV1 199 and SPV1200 have the same genotype with TGL3 deletion but are derived from separate lines, SPV 968 and SPV 969, respectively (Figure 21). The observed reduction is not only due to the presence of FAT1 loss. This is because SPV 956, which is the parent strain of SPV 1056 and SPV 1199, contains the lack of FAT 1 and exhibits higher average specific productivity at 32 ° C. (FIG. 22B, FIG. 24B). The same linear dependence of specific productivity on desaturase copy number was observed at 32 ° C. (FIG. 24B). The gradient increased to about 2.5 (mg / L-OD-h-copy number).

[00657] Z11-16 acid titer
[00658] The highest Z11-16 acid titer was continuously observed at the last 48 hours of sampling. Maximum specific productivity roughly correlates with the 48 hour titer (FIG. 25A). Data is scattered by fluctuations in how long productivity lasts.

[00659] Three of the SPV 969 strains, including the absence of TGL3, remained productive longer and had higher titers (Figure 22C). SPV 969 produced 5.06 ± 0.17 g / L at 32 ° C. Three H. Strain SPV1058 with the zea desaturase copy consistently maintains productivity, with the highest titer observed, 5.79 ± 0.01 g / L at 28 ° C., 6.1 ± 0.08 g / L at 32 ° C. Produced. SPV 1059, the second descendent of SPV 969, contained the loss of both TGL3 and GUT2. When cultured in high glucose medium without glycerol, single replicates of SPV 1059 survived without becoming hypoxic due to seal clogging and reached a titer of 5.39 g / L (material for explanation of seal clogging) And see how).

[00660] The strains SPV 968 and SPV 1056 showed maximal specific productivity similar to the strain in strain SPV 969 at 28 ° C, but the productivity decreased more rapidly, with a 48 hour titer of 2.27 ± 0.07 g / h respectively L and 3.09 ± 0.23 g / L. Productivity remained longer at 32 ° C. and final titers were higher. SPV 968 produced 3.55 ± 0.22 g / L of Z11-16 acid. The SPV 1056 reached a final 48 hour titer of 3.80 ± 0.09 g / L at 32 ° C. The control SPV 459 produced 2.36 ± 0.03 g / L at 28 ° C. and 3.70 ± 0.33 g / L at 32 ° C. (FIG. 22C).

[00661] Selectivity and Lipid Content
[00662] The selectivity at 48 hours was also used to assess the performance of the strains. As defined above, the selectivity is how selectively the biocatalyst produces Z11-16 acid relative to other fatty acid products, and how well the 16 acid substrate is converted to product Show both. The selectivity was high at 32 ° C. for most strains (FIG. 26A). Natural fatty acid titers are consistent and relatively low at both temperatures, primarily due to the sustained conversion of 16 acids to Z11-16 acids. As a result, the selectivity is approximately correlated with Z11-16 acid titer (FIG. 25B). For most strains, the selectivity was relatively constant at 23 and 48 hours (FIG. 25C). At 28 ° C., the second and third generation strains with multiple desaturase copies showed significantly higher selectivity than SPV 459. Maximum selectivity for SPV 1058 and SPV 1059 was observed at about 59% (FIG. 26A). At 32 ° C., the difference in selectivity decreased, with 45% Z11-16 acid for SPV 459 and again 59% for SPV 1058 (FIG. 26A).

[00663] Cell density was measured by OD600 absorbance while moderately obese Y. coli. The lipid fraction of biomass was estimated by converting the absorbance to dry cell weight (DCW) using a coefficient of 0.78 g DCW / L-OD for L. lipolytica cells. The lipid fraction was calculated by dividing the sum of fatty acid titers by this estimate of the dry cell weight concentration. In general, these estimated lipid fractions amounted to 30% to 40% of the total cell weight in 48 hours. SPV 969 and SPV 1058 had the largest lipid fraction at 32 ° C., 44% and 43%, respectively (FIG. 26B). Both SPV 459 and SPV 1056 had lower lipid fractions at 32 ° C. (FIG. 26B). Conversely, SPV 1058 produced slightly higher fractions at 32 ° C. (FIG. 26B).

[00664] The lipid fraction appears to be controlled independently of the other parameters measured, indicating that total lipid storage is regulated independently. This hypothesis is consistent with the observation that SPV 459 takes up 16 acids in experiments with DASGIP fermentors and further converts the 16 acid fraction to Z11-16 acids after first storing the combination of 16 acids and Z11-16 acids. ing. In the Biolector assay, there is no correlation observed between Z11-16 acid productivity and final lipid fraction, indicating that increased flux to Z11-16 acid does not affect lipid storage (Figure 25F). The lipid fraction also does not correlate well with the Z11-16 acid titer for the strains tested (FIG. 25D and FIG. 25E). As mentioned above, the lipid fraction is the same for all strains, and the difference in Z11-16 acid titer is primarily a function of the degree of conversion of 16 acids to Z11-16 acids. A further increase of the Z11-16 acid titer could be achieved by increasing the lipid content of the cells.

[00665] Comparison of performance of high glucose medium and high glycerol medium at 28 ° C (HSD 062)
[00666] In experimental HSD062, the primary high glucose medium was compared to the high glycerol medium used in the batch phase of the current fed-batch fermentation process using SPV 459 (see Materials and Methods for medium composition). The only difference between high glucose and high glycerol media was the concentration of glucose and glycerol co-substrate. High glucose medium contains 120 g / L glucose and 5 g / L glycerol, while high glycerol medium contains 40 g / L glucose and 60 g / L glycerol. The initial growth rate in experiment HSD062 was similar in high glucose and high glycerol media (Figure 27).

[00667] Specific productivity
[00668] Specific productivity changed as a function of strain, medium, and time point. The wells used for the 23 hour sample of SPV 459 in high glucose medium were lost due to seal clogging and accurate measurement of specific productivity could not be made. The specific productivity at 23 hours in high glycerol conditions was lower than that previously observed at high glucose values (2.4 ± 0.1 mg / L-OD-h) (FIG. 28A). In spite of the low initial specific productivity, SPV 459 increased the production of Z11-16 acid later in high glycerol medium. The specific productivity calculated at 41.5 hours was 4.2 ± 0.2 mg / L-OD-h in high glycerol medium and 2.1 ± 0.5 mg / L-OD-h in high glucose medium. There was (Figure 28B).

[00669] As previously observed in high glucose medium, SPV 968 and SPV 1056 had higher specific productivity at the beginning of bioconversion (Figure 28A). Both strains had lower initial specific productivity in high glycerol medium as compared to high glucose medium, but sustained this low productivity up to 41.5 hours (FIG. 28A and FIG. 28B).

[00670] Titer of Z11-16 Acid
[00671] Due to the specific productivity pattern described above, the titer of Z11-16 acid varied with strain and medium. SPV 458 and SPV 459 produced more Z11-16 acid in high glycerol media, but the titers of SPV 968 and SPV 1056 were similar in both media (FIG. 28C).

[00672] Selectivity and Lipid Content
[00673] The dependence of selectivity and lipid content on the medium also varied with the strain. The selectivity of SPV 459 was equivalent in the two media, despite the significant difference in titer of Z11-16 acid (FIG. 28D). The selectivity of SPV 458 was 35% higher in high glycerol medium, while the titer of Z11-16 acid was 56% higher (Figure 28D and Figure 28C). A smaller increase in the selectivity of Z11-16 acid was observed with SPV 968 (17%) and SPV 1056 (6%) (FIG. 28D).

[00674] SPV 459 accumulates higher titers of 16 acids, Z9-18 acids, and Z9Z12-18 acids in high glycerol medium, and the correlation between the selectivity of Z11-16 acid and the titer observed for high glucose medium Was not carried over to high glycerol medium. Strains SPV 968 and SPV 1056 maintained similar selectivity by reducing the accumulation of 16 acids in high glycerol medium, while increasing the titers of Z9-18 and Z9Z12-18 acids (Figure 29).

[00675] The trend in estimated lipid content was consistent with the observations regarding titer and selectivity of Z11-16 acid. The estimated lipid content of SPV 459 and SPV 458 in high glycerol medium was 95% and 56% higher, respectively (FIG. 28E, FIG. 30). SPV 968 and SPV 1056 accumulated similar lipid content in both media.

Abstract
[00676] Taken together, these results support the hypothesis that higher uptake rates of 16 acids in SPV 459 and SPV 458 result in higher intracellular concentrations of substrate for desaturase and consequently higher titers of Z11-16 acids doing. SPV 968 and SPV 1056 did not accumulate increased 16 acid in high glycerol medium, consistent with the uptake rate being independent of glycerol and glucose concentration in these strains. Both SPV 968 and SPV 1056 have FAT1 deficiency, which may explain that they are not affected by media changes. FAT1 has been identified as a lipid body / peroxisomal fatty acid transporter related to lipid mobilization (Dulermo, R., Gamboa-Melendez, H., Ledesma-Amaro, R., Thevenieau, F., & Nicaud, JM (2015 B.) Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 1851 (9): 1202-1217). While not being bound by any one theory, Fat1p may contribute to fatty acid uptake, targeting different membranes in different ways in a medium-dependent manner under certain conditions. Alternatively, this may contribute to the activation of intracellular fatty acids. The increase of natural C18 fatty acids in all strains supports the fast rate of de novo fatty acid synthesis or CoA elongation in high glycerol medium.

[00677] Productivity, maximal titers, and selectivities of major strains grown in primary high glucose medium are shown in Table 17 below. Several important observations can be obtained from the data.

[00678] At 28 ° C, the specific productivity increases with the desaturase copy number. The increase is non-linear and a slight increase is observed with the addition of the third desaturase copy.

[00679] Increasing the incubation temperature from 28 ° C to 32 ° C increases specific productivity in most strains. Exceptions are SPV 1056, SPV 1199, and SPV 1200. These strains are all H.. Has 3 copies of zea desaturase and the lack of FAT1.

[00680] The titer of Z11-16 acid generally increases with the desaturase copy number. The TGL3-deficient strains SPV 969 and SPV 1058 produced maximal titers (5-6 g / L) in 48 hours.

[00681] The titer of Z11-16 acid is higher at 32 ° C. across all strains.

[00682] Selectivity correlates with the titer of Z11-16 acid. The greatest reproducible selectivity was observed with SPV 1056 (0.57 ± 0.02) and SPV 1058 (0.59 ± 0.01).

[00683]

[00684] Experiment HSD062 compared the performance of SPV459, SPV458, SPV968, and SPV1056 in high glucose and high glycerol medium conditions at 28 ° C (Table 18).

[00685] When cultured in high glycerol medium, SPV 458 and SPV 459 have higher total fatty acid titers (7.8 and 10.6 g TFA / L, respectively), Z11-16 acid titers (2.1 and 3.8 g Z11-16 acid / L), and lipid fractions (0.47 and 0.52 g Z11-16 acid / g TFA) were shown.

[00686] SPV 968 and SP 1056 showed higher specific productivity in high glucose medium in the first 23 hours. Overall performance was similar in SPV 1056 in both media, and in SPV 968 high glucose media was better.

[00687]

[00688] The observed specific productivity, estimated specific productivity, maximum titer, and selectivity of all strains grown in primary high glucose medium are shown in Table 19 below.

[00689]

Conclusion
[00690] In High Glucose Medium:
[00691] Additional copies of Z11 desaturase increased the specific productivity of Z11-16 acid.
[00692] Lack of lipid metabolism increased the titer of Z11-16 acid.

[00693] The combination of the additional desaturase copy in SPV 1058 and the lack of the intracellular lipase TGL3 is approximately 65 of the titer of Z11-16 acid compared to SPV 459 (3.7 g / L, 3.7 mg / L-OD-h). % Increase (6.1 g / L) and an increase of about 55% in specific productivity (5.7 mg / L-OD-h).

[00694] The effect of changing to high glycerol media is strain dependent. SPV 459 and SPV 458 produce significantly higher Z11-16 acid titers in high glycerol medium compared to high glucose medium on a 700 μl scale. SPV1056 functions similarly in both media.

[00695] The SPV 1056 and / or SPV 1058 will serve as a second generation strain for process development.

[00696] Co-substrate composition is taken into account in the development of second generation processes.

[00697] Recombinant acyltransferase expression is tested to increase fatty acid storage (gTFA / gDCW). The current strain shows 40-50% fatty acids in the Biolector 700 μl assay.

Materials and methods
[00698] Biolector strain screening bioconversion
[00699] Y. The L. lipolytica strain was inoculated from a YPD agar patch and grown in 2 ml YPD seed culture at 28 ° C., 1000 rpm for 16 hours to an OD 600 = 10 (Infors plate incubator). The seed culture was pelleted and concentrated to OD600 = 80 in fresh YPD. Fresh media were seeded on M2Plabs 48-well flower plates with 1.5-2.5% inoculation. Generally, a volume of 700 μl was used, except for experiment HSD061 where both 600 μl and 700 μl volumes were tested at 32 ° C. GUT2 lacking and control strains generally used high glucose medium except for experiment HSD043 where high glucose medium without glycerol and experiment HSD062 where strains were cultured in high glycerol medium (Table 20 below). After growth for 7 hours at 28 ° C. or 32 ° C., 1500 rpm in a Biolector, remove the plate, add 8.4 μl of 37 ° C. methylpalmitate as a liquid to each well, reseal the plate and return to the Biolector The Twenty-three hours after inoculation, 250 μl samples were removed from rows A and B and transferred to glass crimp top GC vials. The sample vials were frozen and stored at -80 ° C. After sampling, an additional 8.4 μl of 37 ° C. methyl palmitate was added to each well. The flower plate seal was returned and incubated for another 8 hours (at 31 hours). It was then sampled from columns C and D as described for columns A and B. The plate was then incubated for 17 hours (at 48 hours) and samples were removed from rows E and F. Cell density was measured on a Tecan M200 Pro plate reader at each sampling. HSD 062 was performed as described above except that no samples were removed at 31 hours and 4 replicate samples were removed from rows C to F 41.5 hours after inoculation.

[00700] Dissolved oxygen (DO) was monitored using oxygen sensitive optodes on Biolector plates in all experiments. Several experimental replicates were lost due to the low oxygen conditions with biofilms that clog plate seals (see, eg, FIGS. 31A-31E). Based on DO data, clogging can vary and change the growth rate and the degree of perturbation of production of Z11-16 acid. Only repeated data with sustained oxygen transfer were used for analysis. Plots of all data are shown in FIGS. 31A-31E.

[00701]

[00702] GC sample processing-lyophilized sample
[00703] 250 μL of culture was lyophilized in an open glass crimp top vial for at least 3 hours. 500 μL of TMSH was added to the vial and sealed with a crimp cap. The vials were placed in a rack and placed in a 28 ° C. plate shaker for 2 hours at 250 rpm. After mixing these dry cells with the derivatization reagent, the vials were incubated in a heat block at 85 ° C. for 1 hour to lyse cell membranes. Finally, the liquid portion of the methylated sample was transferred to a clean GC vial with a glass insert to prevent solid debris from clogging the column during GC analysis. PROVIVI_002_DUAL_EXTRAWASH_1. The samples were measured by GC-FID using method M. The parameters are shown below.

[00704] Front Entrance / Detector System 6890 GC, ChemStation Rev. B. 03.02 (341)
Column J & W DB-23 30m x 25mm x 25μm
Runtime = 14.4 min inlet heater = 240 ° C., pressure = 9.0 psi, the total flow rate {H2} = 36.2 mL / min Carrier H ₂ @ 1.0 mL / min, 9.0 psi, 35 cm / sec signal data rate = 2Hz / 0.1 minute oven 150 ° C, 1 minute
Gradient 12 ° C / min to 220 ° C, 3 minutes holding
Gradient 35 ° C / min to 240 ° C, holding 4 minutes
Equilibration time: 2 minutes injection split, 240 ° C
Split ratio-30: 1; 29.1 mL / min Detector FID, 240 ° C
H ₂ @ 35.0 mL / min, air @ 350 mL / min
Electrometer {Lit offset} @ 2.0 pA
Sample injection volume = 1 μL

[00705] Back entrance / detector system 6890 GC, ChemStation Rev. B. 03.02 (341)
Column J & W DB-23 30m x 25mm x 25μm
Runtime = 14.4 minutes inlet Heater = 240 ° C, pressure = 9.8 psi, total flow {H2} = 40.1 mL / min Carrier H ₂ @ 1.1 mL / min, 9.8 psi, 38 cm / sec Signal Data rate = 2Hz / 0.1 minute oven 150 ° C, 1 minute
Gradient 12 ° C / min to 220 ° C, 3 minutes holding
Gradient 35 ° C / min to 240 ° C, holding 4 minutes
Equilibration time: 2 minutes injection split, 240 ° C
Split ratio-30: 1; 32.3 mL / min Detector FID, 240 ° C
H ₂ @ 35.0 mL / min, air @ 350 mL / min
Electrometer {Lit offset} @ 2.0 pA
Sample injection volume = 1 μL

[00706] TMSH: trimethylsulfonium hydroxide (0.2 mol / L in methanol)-VWR TCT 1576-025 ML

[00707]

[00708] The above detailed description is presented for clarity of understanding only and modifications are obvious to one of ordinary skill in the art, and therefore unnecessary limitations should not be understood therefrom.

[00709] Although the present disclosure has been described in connection with specific embodiments thereof, further modifications are possible, and the present application generally follows the principles of the present disclosure and is known or within the scope of the art to which the present disclosure relates. Any variant, use, or use of the present disclosure, including the essential features described above and departures from the present disclosure, which may apply to the claims appended below, included in conventional implementations. It will be understood that it is intended to encompass adaptation.

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However, reference to any reference, article, publication, patent, patent publication, and patent application cited herein constitutes prior art in which it is useful or in any country in the world. It does not admit or suggest any form or form of universal general knowledge, nor should it be received as such.

Claims (112)

A method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties, comprising
Obtaining the one or more unsaturated lipid moieties from one or more naturally occurring organisms; and reducing the one or more unsaturated lipid moieties, wherein the one reducing Or producing a plurality of fatty alcohols and / or one or more fatty aldehydes. A method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties, comprising
Obtaining said one or more unsaturated lipid moieties from one or more naturally occurring organisms;
Chemically converting the one or more unsaturated lipid moieties to one or more free fatty acids (FFAs);
Esterifying the one or more FFAs to one or more fatty acid alkyl esters (FAAEs); and reducing the one or more FFAs and / or the one or more FAAEs. A method comprising the step of: producing one or more fatty alcohols and / or one or more fatty aldehydes by said reduction. A method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties, comprising
Obtaining said one or more unsaturated lipid moieties from one or more naturally occurring organisms;
Chemically converting the one or more unsaturated lipid moieties into one or more FAAEs; and reducing the one or more FAAEs, wherein the reduction results in one or more fats. A method comprising the step of producing alcohol and / or one or more fatty aldehydes.   A method according to any one of the preceding claims, wherein the one or more naturally occurring organisms are insects.   A method according to any one of the preceding claims, wherein the one or more naturally occurring organisms are not insects.   6. The method according to any one of the preceding claims, wherein the one or more naturally occurring organisms are selected from the group consisting of plants, algae and bacteria.   7. The method according to claim 6, wherein the plant is derived from a family selected from the group consisting of pokeweed family and buckthorn family.   The above-mentioned plants are Gevuina, Calendula, Limnanthes, Lunaria, Carum, Daucus, Coriandrum, Macadamia, Myristica, Myristica, Licania, Aleurites, Ricinus, Corylus, Kermadecia, Asclepias, Cardwellia 7. The method according to claim 6, comprising a species from the genera Grevillea, Orites, Ziziphus, Hicksbeachia, Hippophae, Ephedra, Plasospermum, Xylomelum and / or Simmondsia.   Said plants are Gevuina avellana, Corylus avellana, Kermadecia sinuata, Asclepias syriaca, Cardwellia sublimis, Grevillea exul var. 9. A method according to claim 8, selected from the group consisting of rubiginosa, Orites diversifolius, Orites revoluta, Ziziphus jujube, Hicksbeachia pinnatifolia, and Grevillea decora.   7. The method of claim 6, wherein the algae comprises green algae.   11. The method of claim 10, wherein the green algae comprises a species from the genus Pediastrum.   11. The method of claim 10, wherein the green algae comprises Pediastrum simplex.   7. The method of claim 6, wherein the bacteria comprises gram negative bacteria.   14. The method of claim 13, wherein the gram negative bacteria comprises a species from the genus Myxococcus.   14. The method of claim 13, wherein the gram negative bacterium comprises Myxococcus xanthus.   7. The method of claim 6, wherein the bacteria comprises cyanobacteria.   17. The method of claim 16, wherein the cyanobacteria comprises a species from the genus Synechococcus.   17. The method of claim 16, wherein the cyanobacteria comprises Synechococcus elongatus. A method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties, comprising
Producing the one or more unsaturated lipid moieties in one or more recombinant microorganisms engineered to include a biosynthetic pathway for the production of one or more unsaturated lipid moieties;
Chemically converting the one or more unsaturated lipid moieties to one or more free fatty acids (FFAs) and / or one or more fatty acid alkyl esters (FAAEs); and said one or more than one Reducing the FFA and / or the one or more FAAEs, wherein the reduction produces one or more fatty alcohols and / or one or more fatty aldehydes.   20. The method of claim 19, wherein the one or more recombinant microorganisms use one or more carbon sources for the production of the one or more unsaturated lipid moieties.   21. The method of claim 20, wherein the one or more carbon sources comprises sugars, glycerol, ethanol, organic acids, alkanes, and / or fatty acids.   22. The method of claim 21, wherein the one or more carbon sources are selected from the group consisting of alkanes and fatty acids.   23. The method according to any one of claims 19-22, wherein the one or more recombinant microorganisms have high resistance to alkanes and / or fatty acids.   24. The method of any one of claims 19-23, wherein the one or more recombinant microorganisms accumulate lipids.   25. The method of any one of claims 19-24, wherein the one or more recombinant microorganisms comprise one or more bacteria and / or fungi.   26. The method of any one of claims 19-25, wherein the one or more fungi are oleaginous yeast.   27. The method of claim 26, wherein the oleaginous yeast comprises species from the genus Yarrowia and / or Candida.   27. The method according to claim 26, wherein the oleaginous yeast comprises Yarrowia lipolytica and / or Candida viswanathii (Candida tropicalis).   The one or more recombinant microorganisms may comprise one or more desaturases, one or more acyl CoA oxidases, one or more acyl glycerol lipases or sterol ester esterases, one or more flavoprotein pyridine nucleotide cytochromes Reductase, one or more elongases, one or more thioesterases, one or more glycerol-3-phosphate acyltransferases, one or more lysophosphatidic acid acyltransferases, one or more diacylglycerol acylases 29. Any one of claims 19-28, comprising one or more exogenous biosynthetic enzymes selected from transferase and / or one or more lipid binding proteins. The method according to one paragraph or.   30. The method of claim 29, wherein the one or more desaturases are selected from the group consisting of fatty acyl CoA desaturases and fatty acyl ACP desaturases.   30. The method of claim 29, wherein the one or more flavoprotein pyridine nucleotide cytochrome reductase is selected from the group consisting of cytochrome b5 reductase and NADPH dependent cytochrome P450 reductase.   30. The method of claim 29, wherein the one or more elongases are selected from the group consisting of ELO1, ELO2 and ELO3, or any combination thereof.   30. The method of claim 29, wherein the one or more thioesterases are selected from the group consisting of acyl ACP thioesterases and acyl CoA thioesterases.   30. The method of claim 29, wherein the one or more glycerol-3-phosphate acyltransferase is dual glycerol-3-phosphate O-acyltransferase / dihydroxyacetone phosphate acyltransferase.   30. The method of claim 29, wherein the one or more lysophosphatidic acid acyltransferases are selected from the group consisting of SLC1, ALE1 and LOA1, or any combination thereof.   30. The method of claim 29, wherein the one or more diacylglycerol acyltransferases are selected from the group consisting of acyl CoA-dependent acyltransferases and phospholipids: diacylglycerol acyltransferases.   30. The method of claim 29, wherein the one or more lipid binding proteins are sterol carrier protein 2 (SCP2).   The one or more exogenous biosynthetic enzymes may be, for example, Saccharomyces, Yarrowia, Candida, Spodoptera, Helicoverpa, Ostrinia, Amyelois, Lobesia, Cydia, Grapholita, Lampronia, Sesamia, Plodia, Bombyx, Photenix, Rattus, Arabidopsis, and Bicluslut 38. The method according to any one of claims 29 to 37, which is from a genus selected from the group consisting of   Said one or more exogenous biosynthetic enzymes are Saccharomyces cerevisiae, Yarrowia lipolytica, Candida albicans, Candida tropicalis / Candida viswanathii, Spodoptera littoralis, Hedocoverpa zea, Helicoverpa armifoli ate. capitella, Grapholita molesta, Ostrinia scapulalis, Ostrinia furnacalis, Ostrinia A species selected from the group consisting of the group consisting of nubilalis, Ostrinia latipennis, Ostrinia ovalipennis, Plodia interpunctella, Sesamia inferens, Bombyx mori, Phoenix dactylifera, Rattus norvegicus, Arabidopsis thaliana, Plutella xylostella and Bicyclus annana 38 Or the method described in one item.   The one or more recombinant microorganisms lack, destroy, one or more endogenous proteins in a pathway that competes with the biosynthetic pathway for the production of one or more unsaturated lipid moieties. 40. A method according to any one of claims 19 to 39, which is further engineered to mutate and / or reduce its activity.   Such that the recombinant microorganism lacks, destroys, mutates and / or reduces the activity of one or more endogenous proteins involved in one or more undesired fatty acid elongation pathways 41. The method of claim 40, wherein the method is being operated.   The one or more endogenous proteins involved in one or more undesired fatty acid elongation pathways may be β-ketoacyl-CoA reductase, β-hydroxyacyl-CoA dehydratase, enoyl-CoA reductase, or any of these 42. The method of claim 41, wherein the method is selected from the group consisting of combinations of   The recombinant microorganism lacks, destroys, mutates, and / or has the activity of one or more endogenous proteins involved in one or more fat body storage and / or recycling pathways 41. The method of claim 40, wherein the method is operated to reduce.   Said one or more endogenous proteins involved in one or more fat body storage and / or recycling pathways may be triacylglycerol lipases, lysophosphatidic acid acyltransferases, diacylglycerol acyltransferases, acyltransferases, sterilys. 44. The method of claim 43, wherein the method is selected from the group consisting of ester hydrolase, acyl-CoA synthetase, or any combination thereof.   The recombinant microorganism is engineered to lack, destroy, mutate, and / or reduce the activity of one or more endogenous proteins involved in one or more fatty acid degradation pathways 41. The method of claim 40.   46. The method of claim 45, wherein the fatty acid degradation pathway is a beta oxidation pathway.   The one or more endogenous proteins are selected from the group consisting of acyl-CoA oxidase, multifunctional enoyl-CoA hydratase-β-hydroxyacyl-CoA dehydrogenase, β-ketoacyl-CoA thiolase, or any combination thereof 47. The method of claim 46.   46. The method of claim 45, wherein the fatty acid degradation pathway is an omega oxidation pathway.   50. The method according to claim 48, wherein said one or more endogenous proteins are selected from the group consisting of monooxygenase (CYP 52), fatty alcohol oxidase, fatty alcohol dehydrogenase, alcohol dehydrogenase, fatty aldehyde dehydrogenase, or any combination thereof. Method described.   Claim 50. The recombinant microorganism is engineered to lack, destroy, mutate, and / or reduce its activity in one or more endogenous proteins involved in peroxisomal biogenesis. 40. The method according to 40.   The one or more endogenous proteins involved in peroxisomal biogenesis are PEX1, PEX2, PEX3, PEX4, PEX5, PEX7, PEX8, PEX10, PEX11, PEX12, PEX12, PEX13, PEX14, PEX15, PEX17, PEX18 51. The method of claim 50, selected from the group consisting of PEX19, PEX21, PEX22, PEX25, PEX28, PEX28, PEX29, PEX30, PEX31, PEX32 and PEX34, or any combination thereof.   52. The method of any one of claims 40-51, wherein the one or more endogenous proteins are from a genus selected from the group consisting of Saccharomyces, Yarrowia and Candida.   53. The any one of claims 40-52, wherein said one or more endogenous proteins are from a species selected from the group consisting of Saccharomyces cerevisiae, Yarrowia lipolytica, Candida albicans and Candida tropicalis / Candida viswanathii. Method.   54. The method of any of claims 19-53, wherein the one or more recombinant microorganisms are further engineered to increase intracellular levels of coenzyme.   55. The method of claim 54, wherein the coenzyme is NADH and / or NADPH.   The one or more recombinant microorganisms are glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase, transketolase, ribose-5-phosphate ketolisomerase, Selected from the group consisting of D-ribulose-5-phosphate 3-epimerase, NADP + dependent isocitrate dehydrogenase, NAD + or NADP + dependent malate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, transhydrogenase or any combination thereof 56. The method of claim 54 or 55, further manipulated to express one or more endogenous or exogenous proteins.   57. The method of claim 56, wherein the expression of one or more endogenous or exogenous protein is linked to complementation of a co-substrate.   The one or more recombinant microorganisms are glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase, transketolase, ribose-5-phosphate ketolisomerase, D-ribulose-5-phosphate 3-epimerase, or any combination thereof, lacks, destroys, mutates, and / or their activity of one or more endogenous proteins 56. The method of claim 54 or 55, further operating to reduce.   The one or more unsaturated lipid moieties include (Z) -3-hexenoic acid, (Z) -3-nonenoic acid, (Z) -5-decenoic acid, (E) -5-decenoic acid, (Z) ) -7-dodecenoic acid, (E) -8-dodecenoic acid, (Z) -8-dodecenoic acid, (Z) -9-dodecenoic acid, (E, E) -8,10-dodecadienoic acid, (7E, 9Z) -dodecadienoic acid, (Z) -9-tetradecenoic acid, (Z) -11-tetradecenoic acid, (E) -11-tetradecenoic acid, (Z) -7-hexadecenoic acid, (Z) -9-hexadecenoic acid (Z) -11-hexadecenoic acid, (Z, Z) -11, 13-hexadecadienoic acid, (11 Z, 13 E) -hexadecadienoic acid, (9 Z, 11 E) -hexadecadienoic acid, (Z) −1 1-octadecenoic acid, (Z) 13-octadecenoic acid, (Z, Z, Z, Z Z) -3,6,9,12,15- Trichoderma Sa penta-enoic acid, or any combinations thereof, The method according to any one of claims 1-58.   The one or more unsaturated lipid moieties may be (Z) -9-tetradecenoic acid (Z9-14: COOH), (E) -11-tetradecenoic acid (E11-14: COOH), (Z) -11-. Tetradecenoic acid (Z11-14: COOH), (Z) -9-hexadecenoic acid (Z9-16: COOH), (Z) -11-hexadecenoic acid (Z11-16: COOH), (Z) -13-octadecenoic acid 59. The method of any one of claims 1-58, wherein the method is selected from the group consisting of (Z13-18: COOH) and (Z) -11-octadecenoic acid (Z11-18: COOH).   61. Any one of the preceding claims, wherein said one or more fatty alcohols and / or one or more fatty aldehydes comprise one or more insect pheromone, a fragrance, a flavor and / or a polymer intermediate. The method described in.   62. The method of claim 61, wherein the one or more fatty alcohols and / or one or more fatty aldehydes comprises one or more insect pheromone.   63. A recombinant microorganism used in the method of any one of claims 19 to 62 for the production of unsaturated lipid moieties.   64. A method of producing the recombinant microorganism of claim 63. Chemically converting the one or more unsaturated lipid moieties to one or more FFAs and / or one or more FAAEs;
Isolating the one or more unsaturated lipid moieties,
Optionally, concentrating one or more unsaturated lipid moieties having a specific chain length, and treating said one or more unsaturated lipid moieties to produce a fatty acid derivative 63. The method of any one of claims 2-62, further comprising   66. The method of claim 65, wherein treating the one or more unsaturated lipid moieties comprises saponifying the one or more unsaturated lipid moieties to one or more FFAs.   66. The method of claim 65, wherein treating the one or more unsaturated lipid moieties comprises esterification of the one or more unsaturated lipid moieties to one or more FAAEs.   66. The method of claim 65, wherein the optional step of concentrating one or more unsaturated lipid moieties having a particular chain length comprises a separation method.   69. The method of claim 68, wherein the separation method comprises distillation, chromatography and / or distillation.   Reducing the one or more unsaturated lipid moieties, the one or more FFAs and / or the one or more FAAEs to one or more fatty alcohols and / or one or more fatty aldehydes Contacting the one or more unsaturated lipid moieties, the one or more FFAs and / or the one or more FAAEs with one or more stoichiometric reducing agents. 72. The method according to any one of Items 1 to 69.   Said one or more stoichiometric reducing agents comprise sodium bis (2-methoxyethoxy) aluminum hydride Vitride (Red-Al, Vitride, SMEAH) and / or hydrogenated diisobutylaluminum (DIBAL). Method according to 70.   The step of reducing the one or more unsaturated lipid moieties, the one or more FFAs and / or the one or more FAAEs includes a bulky cyclic nitrogen Lewis base to modify the reducing agent 72. The method of claim 70 or 71.   73. The method of claim 72, wherein the bulky cyclic nitrogen Lewis base provides the selectivity of the reduction step for the production of one or more fatty aldehydes.   The step of reducing the one or more unsaturated lipid moieties, the one or more FFAs and / or the one or more FAAEs to one or more fatty alcohols is one or more unsaturated ones. 70. A method according to any one of the preceding claims, comprising contacting a lipid moiety, said one or more FFAs and / or said one or more FAMEs with one or more transition metal catalysts. .   75. The method of claim 74, wherein the one or more transition metal catalysts comprise one or more Group VIII catalysts.   76. The method according to any one of claims 1-70 or 73-75, wherein the fatty alcohol produced is further oxidized to a fatty aldehyde by an oxidation step.   77. The method of claim 76, wherein the oxidation step comprises one or more partial oxidation methods.   78. The method of claim 77, wherein the one or more partial oxidation methods comprise NaOCI / TEMPO.   77. The method of claim 76, wherein the oxidation step comprises copper catalyzed aerobic oxidation.   80. A method according to any one of the preceding claims, wherein the one or more fatty alcohols and / or one or more fatty aldehydes are further concentrated by a separation procedure.   81. A composition produced by the method of any one of claims 1-80.   30. The method of claim 29, wherein the one or more desaturases comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-24.   30. The method of claim 29, wherein the one or more desaturases comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 25-38.   30. The method of claim 29, wherein the one or more acyl CoA oxidases are selected from Table 3a.   30. The method of claim 29, wherein the one or more acyl glycerol lipase or sterol ester esterase is selected from Table 3b.   The said acyltransferase is Y.. lipolytica YALI0 C00209 g, Y. lipolytica YALI 0 E 18964 g, Y. lipolytica YALI 0 F 19514 g, Y. lipolytica YALI 0 C 14014 g, Y. lipolytica YALI 0 E 16 797 g, Y. lipolytica YALI 0 E 327 69 g, and Y. lipolytica YALI 0 D 0 79 86 g, S. S. cerevisiae YBL011 w, S.I. S. cerevisiae YDL052 c, S.I. S. cerevisiae YOR 175 C, S.I. S. cerevisiae YPR139C, S.I. S. cerevisiae YNR 008 w, and S. S. cerevisiae YOR245c, as well as Candida I 503_ 025 77, Candida CTRG_ 02630, Candida CaO 19. 250, Candida Ca O 19. 7881, Candida CT RG _ 02 437, Candida Ca O 19. 1881, Candida Ca O 19 9. 437, Candida CT RG 01 068, Candida Ca O 19 ci. Selected from the group consisting of CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209 45. The method of claim 44, wherein:   Said acyl-CoA oxidase is Y.1. lipolytica POX1 (YALI0E32835 g), Y.1. lipolytica POX2 (YALI0F10857g), Y.1. lipolytica POX3 (YALI0D24750 g), Y.1. lipolytica POX 4 (YALI0E27654 g), Y.1. lipolytica POX5 (YALI0 C23859 g), Y.1. lipolytica POX 6 (YALI0E06567 g); S. cerevisiae POX1 (YGL205W); Candida POX2 (CaO 19.1655, CaO 19.9224, CTRG_02374, M18259), Candida POX4 (CaO 19.1652, CaO 19.9221, CTRG_02377, M12160), and Candida POX5 (CaO 19.5723, CaO19.13, 146) 48. The method of claim 47, wherein the method is selected from the group consisting of CTRG 02721, M12161).   20. The method of claim 19, further comprising chemically converting the one or more fatty alcohols to one or more corresponding fatty acetate esters.   89. The process of chemically converting the one or more fatty alcohols to one or more corresponding fatty acetate esters comprises contacting the one or more fatty alcohols with acetic anhydride. Method described.   90. The method of claim 88 or 89, wherein the one or more fatty acetate esters are further concentrated by a separation procedure.   91. The method of any one of claims 1-80 or 82-90, wherein said FAAE comprises fatty acid methyl ester (FAME). A method of producing one or more fatty alcohols and / or one or more fatty aldehydes from one or more unsaturated lipid moieties, comprising
Fat with 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NO: 1, 32, 32, 34, 36-38 for the production of one or more unsaturated lipid moieties Producing said one or more unsaturated lipid moieties in a Yarrowia lipolytica microorganism engineered to contain at least one nucleic acid molecule encoding an acyl desaturase,
Chemically converting the one or more unsaturated lipid moieties to one or more fatty acid alkyl esters (FAAEs); and reducing the one or more FAAEs by the reduction 1 A method comprising the step of producing one or more fatty alcohols and / or one or more fatty aldehydes. Producing the one or more unsaturated lipid moieties,
At least one nucleic acid molecule encoding a fatty acyl desaturase having 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NO: 1, 32, 32, 34, 36-38 to Yarrowia lipolytica microorganisms Furthermore, cultivating the engineered Yarrowia lipolytica microorganism in a culture medium comprising introducing or expressing; and a feedstock providing a carbon source suitable for the production of said one or more unsaturated lipid moieties. 93. The method of claim 92, comprising.   One or more endogenous that the Yarrowia lipolytica microorganism functions in lipid mobilization and / or catalyzes a reaction in a pathway that competes with the biosynthetic pathway for the production of the one or more unsaturated lipid moieties 93. The method of claim 92, comprising the loss, destruction, mutation, and / or reduction of the activity of the enzyme. The said Yarrowia lipolytica microorganism is
(I) one or a plurality of acyl-coenzymes selected from the group consisting of YALI0E32835 g (POX1), YALI0F10857 g (POX2), YALI0D24750 g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6);
(Ii) YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH07) One or more (fatty) alcohol dehydrogenases;
(Iii) (Fat) alcohol oxidase YALI 0 B 14014 g (FAO 1);
(Iv) fatty acyl CoA synthetase / fatty acid transporter YALI0E16016 g (FAT1); and (v) intracellular triacylglycerol lipase YALI0D 17534 g (TGL3)
93. The method of claim 92, comprising loss, disruption, mutation, and / or reduction of the activity of one or more endogenous enzymes selected from.   93. The method of claim 92, wherein one or more endogenous acyltransferase genes are replaced with one or more variants of heterologous acyltransferase genes selected from Table 3c.   The one or more unsaturated lipid moieties include (Z) -3-hexenoic acid, (Z) -3-nonenoic acid, (Z) -5-decenoic acid, (E) -5-decenoic acid, (Z) ) -7-dodecenoic acid, (E) -8-dodecenoic acid, (Z) -8-dodecenoic acid, (Z) -9-dodecenoic acid, (E, E) -8,10-dodecadienoic acid, (7E, 9Z) -dodecadienoic acid, (Z) -9-tetradecenoic acid, (Z) -11-tetradecenoic acid, (E) -11-tetradecenoic acid, (Z) -7-hexadecenoic acid, (Z) -9-hexadecenoic acid (Z) -11-hexadecenoic acid, (Z, Z) -11, 13-hexadecadienoic acid, (11 Z, 13 E) -hexadecadienoic acid, (9 Z, 11 E) -hexadecadienoic acid, (Z) −1 1-octadecenoic acid, (Z) 13-octadecenoic acid, (Z, Z, Z, Z Z) -3,6,9,12,15- Trichoderma Sa penta-enoic acid, or is selected from the group consisting of any combination, method of claim 92. Chemically converting the one or more unsaturated lipid moieties to one or more FAAE,
Isolating the one or more unsaturated lipid moieties;
Optionally enriching one or more unsaturated lipid moieties having a particular chain length; and esterifying and / or transesterification said one or more unsaturated lipid moieties to one or more FAAEs 93. The method of claim 92, further comprising the step of   99. The method of claim 98, wherein the optional step of concentrating one or more unsaturated lipid moieties having a particular chain length comprises a separation method.   100. The method of claim 99, wherein the separation method comprises distillation, chromatography and / or distillation.   The step of reducing said one or more FAAEs to one or more fatty alcohols and / or one or more fatty aldehydes comprises one or more stoichiometric reducing agents of said one or more FAAEs 93. The method of claim 92, comprising contacting with.   Said one or more stoichiometric reductants comprise sodium bis (2-methoxyethoxy) aluminum hydride Vitride (Red-Al, Vitride, SMEAH) and / or diisobutylaluminum hydride (DIBAL). The method described in 101.   93. The method of claim 92, further comprising the step of oxidizing the one or more fatty alcohols to one or more corresponding fatty aldehydes.   104. The method of claim 103, wherein oxidizing comprises contacting the one or more fatty alcohols with NaOCI / TEMPO.   93. The method of claim 92, further comprising chemically converting the one or more fatty alcohols to one or more corresponding fatty acetate esters.   106. The process of chemically converting the one or more fatty alcohols to one or more corresponding fatty acetate esters comprises contacting the one or more fatty alcohols with acetic anhydride. Method described. Oxidizing the one or more fatty alcohols to one or more corresponding fatty aldehydes; and / or chemically converting the one or more fatty alcohols to one or more corresponding fatty acetate esters The step of
Further include
93. The method of claim 92, wherein the one or more fatty alcohols, one or more fatty aldehydes and / or one or more fatty acetate esters are further concentrated by a separation procedure.   93. The method of claim 92, wherein the one or more FAAEs comprise one or more fatty acid methyl esters (FAMEs).   93. The method of claim 92, wherein said fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 41, 42 and 43.   93. The method of claim 92, wherein the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or a desaturase from Trichoplusia ni.   The Yarrowia lipolytica microorganism is a MATA ura3-302 :: SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox 6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh5 Δadh6 Δadh7 Δadh7 A composition produced by the method of claim 92 or claim 107.